SYSTEMS AND METHODS FOR BIOMOLECULAR PRODUCTION

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 139,818, filed on January 21, 2021.BACKGROUND

[0002] Polypeptides, including proteins and / or peptides, are commonly found in a broad range of natural, artificial or synthetic environments, including in vivo and ex vivo environments. Polypeptides may be isolated from these environments for purposes such as research, diagnostics, or commercial usage. Polypeptides may be purified, modified, or otherwise prepared before subsequent usage.

[0003] WO 2017 / 125442 A1 discloses a method for analysing protein variants of a recombinant protein of interest, such as antibodies or Fc-fusion proteins, in a liquid sample of a mammal.SUMMARY

[0004] The presently claimed invention is as defined in the claims.

[0005] The invention provides a method of forming a polypeptide array, comprising: (a) providing a sample comprising sample polypeptides; (b) separating the sample polypeptides from the sample in the presence of separation standard polypeptides, wherein the separation standard polypeptides comprise polypeptides derived from a differing source than the sample polypeptides, and wherein each separation standard polypeptide of the separation standard polypeptides comprises a universal and / or unique tag, wherein the universal tag distinguishes the separation standard polypeptides from other polypeptides in the method and wherein the unique tag distinguishes a separation standard polypeptide from all other separation standard polypeptides in the method, wherein the tag is a peptide sequence; (c) coupling the sample polypeptides to anchoring groups to form sample polypeptide composites, wherein the coupling occurs in the presence of coupling standard polypeptides, wherein the coupling standard polypeptides comprise polypeptides derived from a differing source than the sample polypeptides, and wherein each coupling standard polypeptide of the coupling standard polypeptides comprises a universal and / or unique tag, wherein the universal tag distinguishes the coupling standard polypeptides from other polypeptides in the method and wherein the unique tag distinguishes a coupling standard polypeptide from all other coupling standard polypeptides in the method, wherein the tag is a peptide sequence; and (d) attaching at least a fraction of the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides to a solid support, whereby each of the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides that is attached to the solid support comprises an address of the polypeptide array.

[0006] In one embodiment, the attaching of step (d) occurs in the presence of attachment standard polypeptides, whereby each of the attachment standards that is attached to the solid support comprises an address of the polypeptide array.

[0007] In one embodiment, the attachment standard polypeptides comprise attachment standard polypeptide composites, wherein each of the attachment standard polypeptide composites comprises an attachment standard polypeptide coupled to an anchoring group.

[0008] In one embodiment, the separation standard polypeptides comprise separation standard polypeptide composites, wherein each of the separation standard polypeptide composites comprises a separation standard polypeptide coupled to an anchoring group.

[0009] In one embodiment, the coupling standard polypeptides comprise coupling standard polypeptide composites, wherein each of the coupling standard polypeptide composites comprises a coupling standard polypeptide coupled to an anchoring group.

[0010] In one embodiment, the separation standard polypeptides, coupling standard polypeptides or attachment standard polypeptides comprise a synthetic polypeptide, a non-natural amino acid or a synthetically modified amino acid.

[0011] In one embodiment, the polypeptide array comprises at least 1 x 10 4< different full-length primary protein structures.

[0012] In one embodiment, the anchoring groups comprise particles configured to couple to the solid support, optionally wherein the particles comprise a nucleic acid, a polypeptide, a polymer, an inorganic nanoparticle, an organic nanoparticle, a structured nucleic acid particle (SNAP), a nucleic acid origami or a nucleic acid nanoball.

[0013] In one embodiment, the method further comprises: (e) detecting the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides at addresses of the polypeptide array.

[0014] In one embodiment, the method further comprises detecting the attachment standard polypeptides at addresses of the polypeptide array.

[0015] In one embodiment, the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides are detected as single polypeptide resolution.

[0016] In one embodiment, the method further comprises quantifying the amount of a particular sample polypeptide species in the sample by comparing (i) a number of the addresses attached to a separation standard polypeptide with (ii) the number of the addresses attached to a sample polypeptide composite that comprise the particular sample polypeptide species.

[0017] In one embodiment, the method further comprises quantifying the amount of a particular sample polypeptide species in the sample by comparing (i) a number of addresses attached to a coupling standard polypeptide with (ii) a number of addresses attached to a sample polypeptide composite that comprise the particular sample polypeptide species.

[0018] In one embodiment, the method further comprises quantifying the amount of a particular sample polypeptide species in the sample by comparing (i) a number of addresses attached to an attachment standard polypeptide with (ii) a number of addresses attached to a sample polypeptide composite that comprise the particular sample polypeptide species.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The invention is defined in the claims. Any of the following Figures that do not fall within the scope of the claims do not form part of the claimed invention and are provided for comparative purposes only.

[0020] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: FIG. 1A depicts a sample preparation decision framework, in accordance with some embodiments. FIG. 1B depicts a sample preparation decision framework, in accordance with some embodiments. FIG. 1C depicts a sample preparation decision framework, in accordance with some embodiments. FIG. 1D depicts a sample preparation decision framework, in accordance with some embodiments. FIG. 2 depicts a flow chart for methods of separating proteins from a biological sample, in accordance with some embodiments of the present invention. FIG. 3 depicts a flow chart for methods of separating proteins from a curated sample, in accordance with some embodiments of the present invention. FIG. 4A illustrates a step of a targeting separation method for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 4B illustrates a step of a targeting separation method for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 4C illustrates a step of a targeting separation method for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 4D illustrates a step of a targeting separation method for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 4E illustrates a step of a targeting separation method for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 4F illustrates a step of a targeting separation method for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 4G illustrates a step of a targeting separation method for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 5A shows a step of utilizing a degradable targeting agent for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 5B shows a step of utilizing a degradable targeting agent for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 5C shows a step of utilizing a degradable targeting agent for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 5D shows a step of utilizing a degradable targeting agent for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 5E shows a step of utilizing a degradable targeting agent for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 5F shows a step of utilizing a degradable targeting agent for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 5G shows a step of utilizing a degradable targeting agent for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 5H shows a step of utilizing a degradable targeting agent for isolating a sample polypeptide fraction, in accordance with some embodiments. FIG. 6A depicts a step of a method for functionalizing sample polypeptides from a biological source, in accordance with some embodiments. FIG. 6B depicts a step of a method for functionalizing sample polypeptides from a biological source, in accordance with some embodiments. FIG. 7A depicts a step of an alternative method for functionalizing sample polypeptides from a biological source, in accordance with some embodiments. FIG. 7B depicts a step of an alternative method for functionalizing sample polypeptides from a biological source, in accordance with some embodiments. FIG. 8A depicts a step of a method for functionalizing sample polypeptides from a curated source, in accordance with some embodiments. FIG. 8B depicts a step of a method for functionalizing sample polypeptides from a curated source, in accordance with some embodiments. FIG. 9A depicts a method for forming a polypeptide composite coupled to a solid support, in accordance with some embodiments. FIG. 9B depicts a method for forming a polypeptide composite coupled to a solid support, in accordance with some embodiments FIG. 10 illustrates the formation of polypeptide composites comprising differing species of anchoring groups, in accordance with some embodiments. FIG. 11A illustrates a method for forming an array of polypeptide composites comprising multiple species of polypeptide composites, in accordance with some embodiments. FIG. 11B illustrates an alternative method for forming an array of polypeptide composites comprising multiple species of polypeptide composites, in accordance with some embodiments FIG. 12 shows a flowchart for forming arrays of polypeptide composites coupled to solid supports, in accordance with some embodiments. FIG. 13 displays HPLC data for a protein conjugate demonstrating the change in absorption profile between the functionalized protein (upper) and non-functionalized protein (lower). FIG. 14A shows the effect of the presence of surfactants and denaturants on the functionalization of hen-egg white lysozyme. FIG. 14B shows the effect of the presence of surfactants and denaturants on the functionalization of myoglobin. FIG. 15A displays HPLC data for the conjugation of Alexa-Fluor 647-labeled Protein A to a DNA origami anchoring group. FIG. 15B displays negative control HPLC data for the conjugation of Protein A to a DNA origami anchoring group with no reactive handle. FIG. 15C displays HPLC data for the conjugation of Alexa-Fluor 488-labeled maltose-binding protein to a DNA origami anchoring group. FIG. 15D displays HPLC data for the conjugation of TAMRA-labeled ubiquitin to a DNA origami anchoring group. FIG. 16 illustrates a schematic view of an anchoring group comprising multiple nucleic acid origami components, in accordance with some embodiments. FIGs 17A, 17B, and 17Cdepict top-down views of polypeptide arrays comprising ordered spatial distributions of standard polypeptides, in accordance with some embodiments. FIG. 17Ddepicts a top-down view of a polypeptide array comprising a random spatial distribution of standard polypeptides, in accordance with some embodiments. DETAILED DESCRIPTION

[0021] The characterization of polypeptide samples derived from natural or engineered systems is often difficult due to the wide or dynamic range of polypeptide abundances found within these systems. For example, a single cell (such as a bacterium or tissue cell) may contain some polypeptides with thousands or even millions of copies and other polypeptides with ten or fewer copies. Consequently, in polypeptide samples with diverse types of polypeptides present, the signal generated by the characterization of the most abundant polypeptides can drown out the signal generated by the least abundant polypeptides. For methods such as mass spectrometry, the analysis of low copy number polypeptides may require collection of enough of the low copy number polypeptide to produce a detectable signal.

[0022] Single molecule approaches to polypeptide characterization provide the ability to detect low copy number polypeptides due to the molecule-by-molecule approach to analysis. Single-molecule approaches to polypeptide characterization, such as affinity binding approaches and Edman degradation sequencing approaches, can provide detectable signal over the full range of polypeptide abundances within a proteome. However, the detection of low copy number polypeptides utilizing a single-molecule approach may be sensitive not only to any detection bias arising from the characterization assay itself, but also detection bias arising due to polypeptide sample preparation methods. For example, if a polypeptide sample preparation method has a known bias against hydrophobic polypeptides, then low copy number hydrophobic polypeptides may be excluded from a sample, thereby limiting or eliminating the characterization of these polypeptides. Moreover, exclusion of polypeptides from a sample can adversely impact the ability to accurately quantify polypeptides in the sample.

[0023] The present disclosure describes methods and systems for the preparation of polypeptide samples for any of a variety of purposes, including, for example, polypeptide assays, such as polypeptide characterization assays or polypeptide quantitation assays. The ability to accurately characterize properties of polypeptides or quantify polypeptides from a complex source, such as a biological system, can be compromised due to modification or degradation of sample polypeptides that occur between the time they are removed from the source and the time they are detected. Complex populations of polypeptides, such as those that are processed when performing proteomic scale preparation and detection methods, can be especially compromised by artifacts arising after the polypeptides are removed from biological systems since polypeptides are capable of modifying and degrading each other, often in ways that create chain reactions due to modifications of one polypeptide that increase its ability to modify other polypeptides. The problem of distinguishing native properties of polypeptides from artifacts introduced during preparation or detection of the polypeptides can be alleviated by methods set forth herein that utilize one or more internal standards, such as polypeptide standards, that provide useful information relevant to characterizing the structure or function of one or more sample polypeptides that are processed in a method set forth herein. In some cases, polypeptide standards may provide information about a polypeptide sample preparation processes or about a method used to detect sample polypeptides. The methods and systems described herein may be useful for generating one or more polypeptide fractions with proteome-scale coverage or targeted subfractions of polypeptides from within a sample comprising a polypeptide fraction.

[0024] Polypeptide standards can be particularly useful for at least four reasons. First, polypeptide standards can include structural features that represent a range of polypeptide structures relative to a sample, a proteome, or a biological system. Polypeptide standards may comprise determined or known amino acid sequences that give rise to unstructured strands or ordered strands, such as alpha helices, beta-pleated sheets, or even larger tertiary structures or motifs. Polypeptide standards may comprise structural features that are either reactive to a particular modification or inert to the modification. For example, a polypeptide standard can include a recognition sequence that is known or expected to be cleaved by a particular protease. The polypeptide standard can be added to a polypeptide sample and the amount of polypeptide standard that is lost or modified while processing the sample can be used to account for absence of sample polypeptides having the protease recognition sequence and that were expected to be present in the sample. Other polypeptide standards can include recognition sequences for other post-translational modifications that may be made to sample polypeptides while being processed in a method set forth herein, and presence or absence of the modifications in sample polypeptides can be evaluated relative to the extent of modifications made to the standards during processing.

[0025] Second, polypeptide standards can include chemical features that encompass a range of possible chemical properties, such as hydrophobicity, hydrophilicity, polarity, surface charge density, and reactivity. For example, polypeptide standards may comprise sequences with consecutive alike amino acids (e.g., RRRRR for a region of high positive-charge density) or consecutive similar amino acids (e.g., IMFIMF for a region of hydrophobicity). A polypeptide standard may provide information on biases in polypeptide preparation processes due to one or more chemical properties (e.g., hydrophobicity, hydrophilicity, charge density, etc.). Polypeptide standards may be composed with polypeptides of varying properties along one or more continua of behavior (e.g., hydrophobic to hydrophilic; polar to nonpolar; positively-charged to neutral to negatively-charged, etc.), thereby providing a measurable standard for biases based upon chemical properties during sample preparation processes.

[0026] Third, polypeptide standards can be particularly useful because they can include unique peptide sequences that provide a tag indicating an associated structure or function. For example, a protease sensitive polypeptide can have a protease recognition sequence and a tag sequence. Detecting presence or absence of the tag sequence in a sample having the polypeptide sequence can provide a convenient means to determine whether or not sample polypeptides that were expected to be in the sample were lost due to proteolysis during sample processing. Many standard polypeptides that are used for a particular sample will have tag sequences that are exogenous to the source from which the sample was derived. This allows the standard polypeptides to be readily distinguished from sample polypeptides. However, some sample sources may include one or more endogenous polypeptides that can be usefully exploited as standard polypeptide(s). For example, so called 'housekeeping proteins' or other high abundance polypeptides from a biological source, can be quantified after processing a sample from the source in order to normalize the yield of lower abundance polypeptides from the sample.

[0027] Fourth, polypeptide standards can be particularly useful because they can be detected using the same techniques used to detect sample polypeptides. For example, polypeptide standards can be attached to addresses on an array that includes addresses attached to sample polypeptides, thereby allowing multiplex detection of standard polypeptides and sample polypeptides together. Moreover, polypeptide standards can be provided to arrays in a segregated or directed fashion, or provided a unique or orthogonal detection label, making them readily detectable during polypeptide assays. Similarly, polypeptide standards can be subjected to mass spectrometry detection schemes used for sample polypeptides with minimal modification to the detection scheme that would have been used for sample polypeptides alone. Polypeptide standards can be subjected to mass spectrometry detection after initial analysis by a multiplex detection method.

[0028] Described herein are improved workflows for the preparation of polypeptide samples. Given the broad range of sources from which polypeptides may be derived, including biological systems (e.g., an organelle, a cell, a tissue, a microbiome) and curated and / or non-biological systems (e.g., industrial effluent, geological samples), the described workflows offer the skilled person an adaptable framework for selecting a sample preparation method that best suits the polypeptide sample of interest and / or the polypeptide assay to be used.

[0029] In an aspect, described herein is a method for forming a polypeptide array comprising providing a polypeptide mixture comprising sample polypeptides and separation standard polypeptides, then separating a plurality of sample polypeptides and a plurality of separation standard polypeptides from the polypeptide mixture to form a separation mixture. The separation mixture may then be combined with coupling standard polypeptides to form a coupling mixture. The coupling mixture may then be coupled to a plurality of anchoring groups, creating polypeptide-anchoring group complexes. Before or after the coupling of the coupling mixture to the anchoring groups, the anchoring groups may be coupled to a solid support at addresses on a surface of the solid support. Consequently, a polypeptide array is formed on the surface of the solid support, comprising sample polypeptides, separation standard polypeptides, and coupling standard polypeptides.

[0030] In alternative configurations of the above described method, the coupling standard polypeptides can be combined with the polypeptide sample before the separation mixture is formed. In yet another alternative configuration of the method, the separation standard polypeptides can be omitted such that the polypeptide array does not include the separation standard polypeptides. Similarly, the coupling standard polypeptides can be omitted such that the polypeptide array does not include the coupling standard polypeptides.

[0031] In another aspect, described herein is a composition comprising a polypeptide array coupled to a solid support. The polypeptide array comprises a plurality of polypeptides, including sample polypeptides, separation standard polypeptides, and coupling standard polypeptides, with each polypeptide in the plurality of polypeptides coupled to an anchoring group. Each anchoring group is coupled to a surface of the solid support to form the polypeptide array. The plurality of sample polypeptides may be characterized as having been coupled to the array with an overall efficiency relative to the amount of sample polypeptides originally present. The overall efficiency may be calculated as a function of a separation efficiency that is a function of the observed plurality of separation standard polypeptides, and / or a coupling efficiency that is a function of the observed plurality of coupling standard polypeptides. Again, the separation standard polypeptides or coupling standard polypeptides can optionally be omitted from the polypeptide array.

[0032] The above described polypeptide array and method for forming a polypeptide array are exemplified with respect to use of separation standard polypeptides and coupling standard polypeptides. The separation standard polypeptides can be useful for characterizing a property of the separation method, characterizing a property of the sample polypeptides and / or quantifying the sample polypeptides. The coupling standard polypeptides can be useful for characterizing a property of the coupling method, characterizing a property of the sample polypeptide-anchoring group complexes and / or quantifying the sample polypeptide-anchoring group complexes. Those skilled in the art will recognize from the teachings of the present disclosure that other types of standard polypeptides can be useful. Standard polypeptides can be introduced at any of a variety of stages of a polypeptide preparation method including, for example, stages set forth herein or known in the art. The standard polypeptides can be useful for characterizing a property of the stage, characterizing a property of sample polypeptides obtained from the stage and / or quantifying sample polypeptides obtained from the stage. For example, a variety of methods set forth herein include a stage of attaching sample polypeptides to a solid support. Attachment standard polypeptides can be combined with a sample, or fraction thereof, prior to or during the attachment stage and the attachment standard polypeptides can be useful for characterizing a property of the attachment stage, characterizing a property of sample polypeptides obtained from the attachment stage and / or quantifying sample polypeptides obtained from the attachment stage.

[0033] It will be readily recognized that the compositions, systems, and methods set forth herein may be readily extended from polypeptide arrays to arrays of other single analytes, such as nucleic acids, polysaccharides, cells, and metabolites.Definitions

[0034] As used herein, the term "sample" refers to a collected substance or material that comprises or is suspected to comprise one or more analytes of interest such as polypeptides. A sample may be modified for purposes such as storage or stability. A sample may have undergone one or more processes that separate or remove unwanted fractions or impurities from the analyte(s) of interest. For example, a fraction is a type of sample. Alternatively, a sample may not have undergone any processes that separates or removes any unwanted fractions or impurities from the analyte(s) of interest. For example, a fluid, tissue or cell is a type of sample, A sample may include biological and / or non-biological components. As used herein, the terms "biological sample" or "biological source" refer to a sample that is derived from a predominantly biological system or organism, such as one or more viral particles, cells (e.g. individualized cells), organelles (e.g. individualized organelles), tissues, bodily fluids, bone, cartilage, and exoskeleton. A biological sample may comprise a majority of biological material on a mass basis, excluding the weight of fluid within the sample. As used herein, the term "curated sample" or "curated source" refer to a sample that is derived from a predominantly non-biological system, such as processed or manufactured materials, rocks, minerals, metals, ceramics, and polymers. Samples obtained from materials that are substantially modified by processing or manufacturing, including materials produced from biological materials (e.g., cotton fabrics) may be classified as curated materials. Curated samples may also include materials associated with industrial processes (e.g., feeds, reagents, mixtures, products, waste, effluents) where biological materials can exist but are expcted to constitute a minority of available sample material on a mass basis. For example, a purified effluent from a biochemical reactor may be a curated sample that is checked for a presence or absence of biological contamination.

[0035] As used herein, the term "fraction" refers to a purified or separated portion of a sample that comprises or is suspected to comprise some, most or all of a particular sample component. For example, a polypeptide fraction may refer to a purified or separated portion of a sample that comprises or is suspected to comprise mostly polypeptides by weight. A fraction may also be characterized with respect to the absence of some, most or all of a particular sample component. For example, a non-polypeptide fraction may refer to a purified or separation portion of a sample that substantially lacks polypeptides or that comprises or is suspected to comprise mostly non-polypeptide substances by weight. Different types of polypeptides can optionally be present in different fractions. For example, a membrane fraction may comprise or be suspected to comprise mostly membrane proteins, and can be substantially devoid of soluble proteins. In another example, a soluble fraction may comprise or be suspected to comprise mostly soluble proteins, and can be substantially devoid of membrane proteins. A sample may undergo one or more processes, such as sample preparation processes set forth herein or known in the art, that cause the sample to be divided into two or more fractions.

[0036] As used herein, the term "polypeptide" refers to a molecule comprising two or more amino acids joined by a peptide bond. A polypeptide may refer to a protein or a peptide. A polypeptide may refer to a naturally-occurring molecule, or an artificial or synthetic molecule. A polypeptide may include one or more non-natural, modified amino acids, or non-amino acid linkers. A polypeptide may be modified naturally or synthetically, such as by post-translational modifications.

[0037] As used herein, the term "sample polypeptide" refers a polypeptide that is contained within a sample, or is obtained by separation from a sample.

[0038] As used herein, the term "separation standard polypeptide" refers to a polypeptide that is used for the purpose of monitoring the performance of a polypeptide separation process. A separation standard polypeptide may be combined with a sample, or fraction thereof, that contains one or more polypeptides that are to be separated from other sample components. A separation standard polypeptide may be derived from the same source as the sample polypeptide (i.e. an endogenous separation standard polypeptide) or from a source that is different from the source of the sample polypeptide (i.e. an exogenous separation standard polypeptide). A separation standard polypeptide may be composed, in part or in whole, of non-natural or abiotic amino acids (D-amino acids), or amino acids joined by a non-amino acid chemical linker.

[0039] As used herein, the term "coupling standard polypeptide" refers to a polypeptide that is used for the purpose of monitoring the performance of a polypeptide coupling process. A coupling standard polypeptide may be combined with a sample, or fraction thereof, that contains one or more polypeptides that participate in a coupling process. A coupling standard polypeptide may be derived from the same source as the sample polypeptide (i.e. an endogenous coupling standard polypeptide) or from a source that is different from the source of the sample polypeptide (i.e. an exogenous coupling standard polypeptide).

[0040] As used herein, the term "protein" may refer to any single- or multi-chain polypeptide molecule. A protein may have a known or unknown biological function or activity. Proteins can include natural, synthetic, modified, and degraded polypeptides.

[0041] As used herein, the term "protein complex" can refer to two or more proteins that are associated or co-localized by covalent or non-covalent bonding between the proteins.

[0042] As used herein, the term "peptide" may refer to any short, single polypeptide chain. A peptide may be no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less than about 5 amino acids in length. A peptide may have a known or unknown biological function or activity. Peptides can include natural, synthetic, modified, or degraded proteins or peptides.

[0043] As used herein, the term "functionalized" refers to any material or substance that has been modified to include a functional group. A functionalized material or substance may be naturally or synthetically functionalized. For example, a polypeptide can be naturally functionalized with a phosphate, oligosaccharide (e.g. glycosyl, glycosylphosphatidylinositol or phosphoglycosyl), nitrosyl, methyl, acetyl, lipid (e.g. glycosyl phosphatidylinositol, myristoyl or prenyl), ubiquitin or other naturally occurring post-translational modification. A functionalized material or substance may be functionalized for any given purpose, including altering chemical properties (e.g., altering hydrophobicity or changing surface charge density) or altering reactivity (e.g. capable of reacting with a moiety or reagent to form a covalent bond to the moiety or reagent).

[0044] As used herein, the term "reactive handle" refers to a pendant, reactive functional group (e.g., activated ester, azide) that is attached to a material or substance. A reactive handle may be covalently or non-covalently attached to a material or substance.

[0045] As used herein, the term "anchoring group" refers to a molecule or particle that serves as an intermediary attaching a protein or peptide to a surface (e.g., a solid support or a microbead). An anchoring group may be covalently or non-covalently attached to a surface and / or a polypeptide. An anchoring group may be a biomolecule, polymer, particle, nanoparticle, or any other entity that is capable of attaching to a surface or polypeptide. In some cases, an anchoring group may be a structured nucleic acid particle.

[0046] As used herein, the term "structured nucleic acid particle" (or "SNAP") refers to a single- or multi-chain polynucleotide molecule having a compacted three-dimensional structure. The compacted three-dimensional structure can optionally have a characteristic tertiary structure. For example, a SNAP can be configured to have an increased number of interactions between regions of a polynucleotide strand, less distance between the regions, increased number of bends in the strand, and / or more acute bends in the strand, as compared to the same nucleic acid molecule in a random coil or other non-structured state. Alternatively or additionally, the compacted three-dimensional structure can optionally have a characteristic quaternary structure. For example, a SNAP can be configured to have an increased number of interactions between polynucleotide strands or less distance between the strands, as compared to the same nucleic acid molecule in a random coil or other non-structured state. In some configurations, the secondary structure (i.e. the helical twist or direction of the polynucleotide strand) of a SNAP can be configured to be more dense than the same nucleic acid molecule in a random coil or other non-structured state. SNAPs may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), and combinations thereof. SNAPs may have naturally-arising or engineered secondary, tertiary, or quaternary structures. Exemplary SNAPs may include nucleic acid nanoballs (e.g. DNA nanoballs), nucleic acid nanotubes (e.g. DNA nanotubes), and nucleic acid origami (e.g. DNA origami). A SNAP may be functionalized to include one or more reactive handles or other moieties.

[0047] As used herein, the term "polypeptide composite" refers to a molecule that is formed by the coupling of a polypeptide to one or more anchoring groups. The coupling between molecules in a polypeptide composite may be covalent or non-covalent. For example, a polypeptide composite may be covalently linked by a covalent bond between a reactive handle on a polypeptide with a reactive handle on an anchoring group. In another example, a polypeptide composite may be non-covalently linked by an interaction such as hybridization between complementary oligonucleotides or a receptor-ligand linkage such as a streptavidin-biotin linkage.

[0048] As used herein, the term "standard," when used in reference to a plurality of analytes (e.g., polypeptides), refers to a composition of one or more molecules that is utilized for characterizing the quality, quantity or other characteristic of a sample or component thereof. A standard may include one or more polypeptides. A standard can be endogenous to a source from which a sample or sample component, such as a sample polypeptide is derived. For example, the endogenous standard can be a polypeptide standard that is derived from the same source from which the sample or sample polypeptide is derived. Accordingly, an endogenous standard polypeptide will have a peptide sequence that is native to, or otherwise found in, the source from which a particular sample or sample polypeptide is derived. Alternatively, a standard can be exogenous to a source from which a sample or sample component, such as a sample polypeptide, is derived. For example, the exogenous standard can be a polypeptide standard that is derived from a source that is different from the source from which the sample or sample polypeptide is derived. Accordingly, an exogenous standard polypeptide will have a peptide sequence that is not found naturally in the source from which a particular sample or sample polypeptide is derived. A standard may include one or more types of polypeptides in known amounts. A standard may further comprise other non-polypeptide components, including small molecule components, that permit observation and / or quantitation of a sample before or after processing of the sample. As used herein, the term "separation standard" may refer to a standard that is utilized to observe the effects of a separation process on a sample, sample fraction (e.g. a polypeptide fraction) or sample component (e.g. a sample polypeptide). As used herein, the term "coupling standard" may refer to a standard that is utilized to observe the effects of a coupling process on a sample, sample fraction (e.g. a polypeptide fraction) or component of a sample (e.g. a sample polypeptide).

[0049] As used herein, the term "species" refers to a molecule with a unique, distinguishable chemical structure. As used herein, the term "polypeptide species" refers to a polypeptide with a unique, distinguishable primary structure. Two polypeptides are of the same species if they possess the same primary structure. Polypeptide variants and isoforms are different species from each other. For example, members of an "anchoring group species" have a unique, distinguishable structure that is common to the members. Anchoring group species may be identified, for example, by common shape and / or conformation, number of coupling sites, or type of coupling sites.

[0050] As used herein, the term "polypeptide diversity" refers to the number of different polypeptide species present within a plurality of polypeptides. The different species can differ with respect to a specified characteristic such as peptide sequence (i.e. polypeptide sequence diversity), length (i.e. polypeptide length diversity), post-translational modification (e.g. polypeptide phosphorylation diversity) or other characteristics set forth herein or known in the art. The number of different polypeptide species present within a plurality of polypeptides can optionally be identified relative to a reference basis, such as the number of polypeptide species in a proteome, within a cell, within a sample, or within a polypeptide fraction. For example, the polypeptide diversity within a polypeptide fraction may refer to the number of species of polypeptides present within the polypeptide fraction relative to the sample from which the polypeptide fraction was derived. The polypeptide diversity may be characterized on a percentage or fractional basis, e.g., 0.1% or 0.001 of all polypeptide species.

[0051] As used herein, the term "click reaction" or "bioorthogonal reaction" refers to single-step, thermodynamically-favorable conjugation reaction utilizing biocompatible reagents. A click reaction may utilize no toxic or biologically incompatible reagents (e.g., acids, bases, heavy metals) or generate no toxic or biologically incompatible byproducts. A click reaction may utilize an aqueous solvent or buffer (e.g., phosphate buffer solution, Tris buffer, saline buffer, MOPS, etc.). A click reaction may be thermodynamically favorable if it has a negative Gibbs free energy of reaction, for example a Gibbs free energy of reaction of less than about - 5 kiloJoules / mole (kJ / mol), -10 kJ / mol, -25 kJ / mol, -50 kJ / mol, -100 kJ / mol, - 200 kJ / mol, -300 kJ / mol, -400 kJ / mol, or less than -500 kJ / mol. Exemplary bioorthogonal and click reactions are described in detail in WO 2019 / 195633A1.

[0052] . Exemplary click reactions may include metal-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted azide-nitrone cycloaddition, strained alkene reactions, thiol-ene reaction, Diels-Alder reaction, inverse electron demand Diels-Alder reaction, [3+2] cycloaddition, [4+1] cycloaddition, nucleophilic substitution, dihydroxylation, thiol-yne reaction, photoclick, nitrone dipole cycloaddition, norbornene cycloaddition, oxanobornadiene cycloaddition, tetrazine ligation, and tetrazole photoclick reactions. Exemplary functional groups or reactive handles utilized to perform click reactions may include alkenes, alkynes, azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines.

[0053] As used herein, the term "array" refers to a population of molecules that is attached to one or more solid supports such that the molecules at one address can be distinguished from molecules at other addresses. An array can include different molecules that are each located at different addresses on a solid support. Alternatively, an array can include separate solid supports each functioning as an address that bears a different molecule, wherein the different molecules can be identified according to the locations of the solid supports on a surface to which the solid supports are attached, or according to the locations of the solid supports in a liquid such as a fluid stream. The molecules of the array can be, for example, nucleic acids such as SNAPs, polypeptides, proteins, peptides, oligopeptides, enzymes, ligands, or receptors such as antibodies, functional fragments of antibodies or aptamers. The addresses of an array can optionally be optically observable and, in some configurations, adjacent addresses can be optically distinguishable when detected using a method or apparatus set forth herein.

[0054] As used herein, the term "address," when used in reference to an array, means a location in an array where a particular molecule is present. An address can contain only a single molecule, or it can contain a population of several molecules of the same species (i.e. an ensemble of the molecules). Alternatively, an address can include a population of molecules that are different species. Addresses of an array are typically discrete. The discrete addresses can be contiguous, or they can have interstitial spaces between each other. An array useful herein can have, for example, addresses that are separated by less than 100 microns, 50 microns, 10 microns, 5 microns, 1 micron, or 0.5 micron. Alternatively or additionally, an array can have addresses that are separated by at least 0.5 micron, 1 micron, 5 microns, 10 microns, 50 microns or 100 microns. The addresses can each have an area of less than 1 square millimeter, 500 square microns, 100 square microns, 25 square microns, 1 square micron or less.

[0055] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquid. The substrate can be non-porous or porous. The substrate can optionally be capable of taking up a liquid (e.g. due to porosity) but will typically be sufficiently rigid that the substrate does not swell substantially when taking up the liquid and does not contract substantially when the liquid is removed by drying. A nonporous solid support is generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon ™< , cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor ™< , silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

[0056] As used herein, the terms "group" and "moiety" are intended to be synonymous when used in reference to the structure of a molecule. The terms refer to a component or part of the molecule. The terms do not necessarily denote the relative size of the component or part compared to the molecule, unless indicated otherwise. The terms do not necessarily denote the relative size of the component or part compared to any other component or part of the molecule, unless indicated otherwise. A group or moiety can contain one or more atom.

[0057] As used herein, the term "sample pretreatment" refers to an in vitro process that alters a sample or a fraction of a sample, or alters a fraction of polypeptides within a sample prior to a sample preparation process (e.g., separation, functionalization, conjugation, etc.). Sample pretreatment processes may include processes that improve the chemical and / or physical properties of a sample or polypeptides within a sample for a subsequent sample preparation process. Sample pretreatment processes may include processes that alter one or more polypeptides within a sample (e.g., drug or drug analog treatments; enzymatic treatments).

[0058] As used herein, the term "epitope" refers to a molecule or part of a molecule, which is recognized by or binds specifically to an affinity reagent. Epitopes may include amino acid sequences that are sequentially adjacent in the primary structure of a protein or amino acids that are structurally adjacent in the secondary, tertiary or quaternary structure of a protein. An epitope can optionally be recognized by or bound to an antibody. However, an epitope need not necessarily be recognized by any antibody, for example, instead being recognized by an aptamer, miniprotein or other probe. An epitope can optionally bind an antibody to elicit an immune response. However, an epitope need not necessarily participate in, nor be capable of, eliciting an immune response.

[0059] As used herein, the term "affinity reagent" refers to a molecule or other substance that is capable of specifically or reproducibly binding to an analyte (e.g. protein, peptide or unique identifier label) or moiety (e.g. post-translational modification of a protein). An affinity reagent can be larger than, smaller than or the same size as the analyte. An affinity reagent may form a reversible or irreversible bond with an analyte. An affinity reagent may bind with an analyte in a covalent or non-covalent manner. Affinity reagents may include reactive affinity reagents, catalytic reaffinity agents (e.g., kinases, proteases, etc.) or non-reactive affinity reagents (e.g., antibodies or fragments thereof). An affinity reagent can be non-reactive and non-catalytic, thereby not permanently altering the chemical structure of an analyte to which it binds. Affinity reagents that can be particularly useful for binding to proteins include, but are not limited to, antibodies or functional fragments thereof (e.g., Fab' fragments, F(ab')2 fragments, single-chain variable fragments (scFv), di-scFv, tri-scFv, or microantibodies), aptamers, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, miniproteins, DARPins, monobodies, nanoCLAMPs, lectins, or functional fragments thereof. The term "affinity agent" is used synonymously with the term "affinity reagent" herein.

[0060] As used herein, the term "single analyte" refers to an analyte (e.g. protein) that is individually manipulated or distinguished from other analytes. A single analyte can be a single molecule (e.g. single protein), a single complex of two or more molecules (e.g. a single protein attached to a structured nucleic acid particle or a single protein attached to an affinity agent), a single particle, or the like. A single analyte may be resolved from other analytes based on, for example, spatial or temporal separation from the other analytes. Accordingly, an analyte can be detected at "single-analyte resolution" which is the detection of, or ability to detect, the analyte on an individual basis, for example, as distinguished from its nearest neighbor in an array. Reference herein to a 'single analyte' in the context of a composition, apparatus or method does not necessarily exclude application of the composition, apparatus or method to multiple single analytes that are manipulated or distinguished individually, unless indicated to the contrary.

[0061] The term "comprising" is intended herein to be open-ended, including not only the recited elements, but further encompassing any additional elements.

[0062] As used herein, the term "each," when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.Polypeptide Sample Preparation Workflows

[0063] Described herein are methods and system for preparing polypeptide fractions for any of a variety of purposes, including, for example, polypeptide assays such as characterizations assays and sequencing assays. The described polypeptide sample preparation workflows may involve one or more fractionation or targeting processes for the isolation of polypeptide fractions. Fractionation processes may include methods that separate samples into two or more fractions (e.g., a polypeptide fraction and a non-polypeptide fraction; soluble polypeptides and membrane polypeptides). Targeting processes may include methods that selectively separate or isolate desired polypeptides from a sample with a broader composition (e.g., capture or pull-down assays). The selection of a sample preparation workflow may be driven, in whole or in part, by the type of polypeptide fraction to be prepared and the intended use of the polypeptide fraction.

[0064] The selection of a polypeptide sample preparation workflow may be influenced by a variety of aligned or competing factors. Factors influencing the choice of sample preparation workflow may include sample type, sample size, sample stability, and mode of use. In some cases, a predominant factor or two or more synergizing factors may influence the selection of a particular workflow. In other cases, two or more competing or offsetting factors may make more than one sample preparation workflow appropriate for generation of a polypeptide fraction.

[0065] The selection of a polypeptide sample preparation workflow may also be influenced by the final form in which a polypeptide sample may be produced. For example, polypeptide fractions produced for a mass spectrometry type analysis may be produced in a solution or loosely adhered to a solid support. In contrast, polypeptide fractions produced for an affinity binding assay may be produced in a more ordered array on a solid support. Consequently, in some cases, it may be advantageous to prepare polypeptide fractions in the presence of a solid support or other medium upon which the polypeptide is to be utilized. Certain separation methods, such as targeting, may be configured for in situ usage (e.g., on-chip devices), while other methods, such as chromatographic fractionation, may be most easily implemented in bench-scale or larger instruments.

[0066] FIGs. 1A - 1D depict examples of how various factors may be weighed to determine a possible sample preparation workflow. FIG. 1A shows an exemplary workflow decision framework when considering sample types. First, it may be determined if a sample is heterogeneous or homogeneous. A heterogeneous sample may comprise multiple phases (e.g., solids, membranes, aqueous liquids, and / or hydrophobic liquids) or multiple types of components (e.g., cells and free proteins). A homogeneous sample may comprise a uniform solution or mixture (e.g., a soluble fraction from a cellular lysate; a wastewater sample). If a sample is heterogeneous, it may next be desired to retain heterogeneity, or to be homogenized into a homogeneous sample. If the sample remains heterogeneous, it may be subjected to a fractionation or targeting process. If the heterogeneous sample is to be divided into multiple polypeptide fractions, appropriate fractionation or targeting processes may be used. In some cases, a single polypeptide fraction may be isolated by a targeting process. If the sample type is homogeneous, it may also undergo an appropriate fractionation or targeting process. If the homogeneous sample is to be divided into multiple polypeptide fractions, appropriate fractionation or targeting processes may be used. In some cases, a single polypeptide fraction may be isolated by a targeting process.

[0067] FIG. 1B shows a workflow decision framework when considering the size or mass scale of a sample. If the sample is large or has a bulk application, a fractionation method may be chosen to isolate one or more polypeptide fractions. If the polypeptide fractions are to be utilized in a polypeptide assay, the fractionation method may occur on external instrumentation rather than an assay platform, such as a flow cell, chip, or cartridge. Alternatively, if the sample is small or will be used in part, a targeting method may be chosen to isolate one or more polypeptide fractions. If the polypeptide fractions are to be utilized in a polypeptide assay, the targeting method may occur directly on or within an assay platform, such as a flow cell, chip, or cartridge.

[0068] FIG. 1C shows a workflow decision framework when considering the time scale or stability of a sample. If a sample is unstable or contains target components that are unstable, a targeting method may be appropriate for rapidly isolating the target components. For example, target polypeptide(s) can be separated from protease enzymes or other enzymes that would post-translationally modify the targeted polypeptide(s). In some cases, a sample may be subjected to a series of two or more sequential targeting or fractionation steps. For example, an unstable polypeptide fraction that has been separated from a sample may be further subjected to additional targeting or fractionation depending upon the utility and stability of a subset of desired polypeptides in the sample. If the polypeptide fractions are to be utilized in a polypeptide assay, the targeting method may occur directly on or within an assay platform, such as a flow cell, chip, or cartridge. If a sample is more stable, it may be prepared in one or more fractions by a slower or more complex fractionation method. If the polypeptide fractions are to be utilized in a polypeptide assay, the fractionation method may occur on external instrumentation rather than an assay platform, such as a flow cell, chip, or cartridge.

[0069] FIG. 1D shows a workflow decision framework when a polypeptide sample is to be analyzed in a polypeptide assay. The workflow chosen may depend upon whether the intended analysis is qualitative or quantitative. For qualitative analysis, the chosen workflow may further be selected based upon whether the analysis is for the full polypeptide content of the sample, or a partial polypeptide fraction of the sample. For full sample analysis, fractionation methods may be preferred for larger quantities of polypeptides. For partial sample analysis, targeting or fractionation methods may be utilized depending upon what quantity of polypeptides are to be analyzed. For quantitative analysis, the chosen workflow may also be selected based upon whether the analysis is for the full polypeptide content of the sample, or a partial polypeptide fraction of the sample. For full sample analysis, fractionation methods may be preferred for large quantities of polypeptides. In some cases, a sample may undergo multiple types of fractionations or repeated cycles of a fractionation method to provide separation and collection of a majority, or even substantially all polypeptides in the sample. For partial sample analysis, targeting methods may be preferred for smaller polypeptide fractions. In some cases, a sample may undergo multiple types of targeting or repeated cycles of a targeting method to provide separation and collection of a majority, or even substantially all targeted polypeptides in the sample.

[0070] A sample may be prepared to form a polypeptide array for any of a variety of purposes, such as a polypeptide assay. In some cases, a method of forming a polypeptide array may comprise one or more of the following steps: 1) providing a sample comprising a plurality of sample polypeptides; 2) separating the plurality of sample polypeptides from the sample in the presence of a plurality of separation standard polypeptides; 3) coupling the plurality of sample polypeptides to a plurality of anchoring groups to form a plurality of sample polypeptide composites, where the coupling occurs in the presence of a plurality of coupling standard polypeptides; and 4) attaching at least a fraction of the sample polypeptide composites of the plurality of sample polypeptide composites to a solid support, whereby each of the sample polypeptide composites that is attached to the solid support comprises an address of a polypeptide array. Optionally, step 4) can further include attaching at least a fraction of the separation standard polypeptides and / or the coupling standard polypeptides to the solid support, whereby each of the separation standard polypeptides and / or the coupling standard polypeptides that is attached to the solid support comprises an address of the polypeptide array. In a further option, step 4) can be carried out in the presence of a plurality of attachment standard polypeptides. The attachment standard polypeptides can be attached to addresses of the polypeptide array.

[0071] One or more of the above steps can be omitted from the sample preparation method. For example, a method of forming a polypeptide array may comprise the steps of: 1) providing a sample comprising a plurality of sample polypeptides; 2) coupling the plurality of sample polypeptides to a plurality of anchoring groups to form a plurality of sample polypeptide composites, where the coupling occurs in the presence of a plurality of coupling standard polypeptides; and 3) attaching at least a fraction of the sample polypeptide composites of the plurality of sample polypeptide composites to a solid support, whereby each of the sample polypeptide composites that is attached to the solid support comprises an address of a polypeptide array. Optionally, step 3) can further include attaching at least a fraction of the coupling standard polypeptides to the solid support, whereby each of the coupling standard polypeptides that is attached to the solid support comprises an address of the polypeptide array. In a further option, step 3) can be carried out in the presence of a plurality of attachment standard polypeptides. The attachment standard polypeptides can be attached to addresses of the polypeptide array.

[0072] The steps set forth above for a method of forming a polypeptide array may be carried out in the order exemplified above or in an alternative order. For example, a method of forming a polypeptide array may comprise the steps of: 1) providing a sample comprising a plurality of sample polypeptides; 2) coupling the plurality of sample polypeptides to a plurality of anchoring groups to form a plurality of sample polypeptide composites, where the coupling occurs in the presence of a plurality of coupling standard polypeptides; 3) separating the plurality of sample polypeptide composites from the sample; and 4) attaching at least a fraction of the sample polypeptide composites of the plurality of sample polypeptide composites to a solid support, whereby each of the sample polypeptide composites that is attached to the solid support comprises an address of a polypeptide array. Step 3) can optionally be carried out in the presence of a plurality of separation standard polypeptides. As a further option, step 4) can further include attaching at least a fraction of the separation standard polypeptides and / or the coupling standard polypeptides to the solid support, whereby each of the separation standard polypeptides and / or the coupling standard polypeptides that is attached to the solid support comprises an address of the polypeptide array.

[0073] Optionally, the plurality of sample polypeptides used in the sample preparation methods set forth above or elsewhere herein may be functionalized with a group that facilitates the coupling of the anchoring group to the sample polypeptide. FIG. 12 illustrates various configurations of a method for forming a polypeptide array. In one configuration, a sample 1200 comprising a plurality of sample polypeptides is provided for the polypeptide array method. In some cases, the plurality of sample polypeptides is separated from the sample by a sample polypeptide separation process 1210. Before or after the sample polypeptide separation process 1210, the plurality of sample polypeptides may be functionalized in a polypeptide functionalization process 1250. After the sample polypeptide separation process 1210 and / or the polypeptide functionalization process 1250, the plurality of sample polypeptides may be coupled to a plurality of anchoring groups 1220 and attached to a solid support 1230 to form a polypeptide array 1240 comprising a plurality of sample polypeptides attached to a solid support by a plurality of anchoring groups. In another configuration, the sample 1200 may be coupled to a plurality of anchoring groups 1220. Before the coupling of the sample 1200 to the plurality of anchoring groups 1220, the sample 1200 may be functionalized in a polypeptide functionalization process 1250. After the coupling of the sample 1200 to the plurality of anchoring groups 1220 and / or the polypeptide functionalization process 1250, the plurality of sample polypeptides may be separated from the sample 1200 in a sample polypeptide separation process 1210. After the sample polypeptide separation process 1210, the plurality of polypeptides may be attached to a solid support 1230 to form a polypeptide array 1240 comprising a plurality of sample polypeptides attached to a solid support by a plurality of anchoring groups. In some cases, the coupling of the plurality of sample polypeptides to the anchoring groups 1220 and the binding of the plurality of the sample polypeptides to the solid support 1230 may occur simultaneously, for example by coupling the plurality of sample polypeptides to a plurality of anchoring groups that are already bound to the solid support. A polypeptide array process may include one or more rinsing processes to remove unbound or loosely adhered molecules from the solid support. The attachment of the anchoring groups to the solid support can be covalent or non-covalent in the above method or in other methods set forth herein.

[0074] The methods of sample preparation disclosed herein may further include the providing of surfactants to one or more processes of the sample preparation method. In some cases, a first sample preparation process (e.g., sample collection, sample storage, sample polypeptide separation, sample polypeptide functionalization, sample polypeptide coupling, or sample polypeptide binding) may utilize a first surfactant and a second sample preparation process may utilize a second surfactant. In some cases, the first surfactant and the second surfactant may be the same species of surfactant. In other cases, the first surfactant and the second surfactant may be a different species of surfactant. By analogy, the skilled person will readily recognize that the use of surfactants may be extended to additional sample preparation processes. A sample preparation process of a sample preparation method may be repeated. In some cases, the sample preparation process may be repeated under varying conditions, such as the presence of differing surfactants between the first cycle of the process and the second cycle of the process.Samples and Sample Collection

[0075] Samples of the present disclosure may initially contain any of a variety of materials derived from an environment of interest. In some cases, a sample may be reasonably expected to contain polypeptides, such as samples derived from living or deceased organisms. In other cases, a sample may be reasonably expected to contain no or minimal polypeptides, such as surface swabs from a clean room or pulverized extraterrestrial rock material. In yet other cases, the presence of polypeptides in a sample may be unknown. A sample of the present disclosure may be collected for any of a variety of purposes, including, but not limited to research, medical diagnostics, forensics, and commercial quality assurance.

[0076] A sample may be derived from an organism or an organism-derived substance or material. A sample may comprise any type of organism, including animals, non-human animals, humans, plants, fungi, bacteria, protozoa, archaea, viruses, or combinations thereof. The organism may be a domesticated, modified, or engineered organism, such as poultry, livestock, genetically-modified crops, non-modified crops, transgenic animals, transgenic plants, or production strains of microorganisms (e.g., E. coli, S. cerevisiae). A sample may comprise a substance derived from an organism, such as an extracellular secretion or debris from a deceased cell. A sample may be collected from a 2D cell culture line, a 3D cell culture line, a plant tissue sample, an animal tissue sample, a non-human animal tissue sample, a fungal tissue sample, a cultured tissue sample, a human patient-derived tissue sample, a veterinary patient-derived tissue sample, a skin or tissue swab, a tissue biopsy sample, a bodily fluid sample (e.g., blood plasma, blood serum, whole blood, urine, cerebrospinal fluid, saliva, semen, vaginal secretions, tears, mucus), a fecal sample, a cellular lysate, a fixed tissue sample (e.g., FFPE), a single-cell organism, a tissue-derived single cell, a secreted sample, an environmental sample, a microbial sample, a microbiome sample, a biofilm sample, or a curated sample.

[0077] A sample may be prepared to form one or more fractions comprising polypeptides. A polypeptide fraction may comprise sample polypeptides derived from a biological source such as an organism set forth above. In some cases, a polypeptide fraction may comprise sample polypeptides from a plurality of differing organisms, such as a microbiome-derived sample or a blood sample, or from a plurality of the same organism such as blood samples from a number of humans pooled into a single sample. A polypeptide fraction may comprise sample polypeptides derived from a curated source. A polypeptide fraction may comprise sample polypeptides derived from a curated source such as a forensic sample, an industrial sample, a consumer product, a geological sample, an archeological sample, a paleontological sample, and an extraterrestrial sample.

[0078] A standard polypeptide may have a peptide sequence that is endogenous to one or more of the organisms or other sources set forth above or elsewhere herein. Alternatively, a standard polypeptide may have a peptide sequence that is exogenous to one, more than one, or all of the organisms or other sources set forth above or elsewhere herein. In some configurations, a standard polypeptide may have a peptide sequence that is not found or not known to be found in any organism.

[0079] A polypeptide fraction formed from a sample may have full proteomic polypeptide coverage, or may contain some subset of a full proteome. For example, a polypeptide sample may provide sufficient amounts of polypeptides to represent the full proteome of a biological source from which the tissue is derived. In another example, a blood plasma sample may only represent the portion of a proteome that is secreted into, or otherwise present in, the bloodstream of an organism from which the sample is derived. A full proteomic sample may be considered a sample that displays the full sequence diversity or content of polypeptides that may be present within a cell, tissue, biological fluid or organism. A partial proteomic sample may be considered a sample that displays a portion of the full sequence diversity or content of polypeptides that may be present within a cell, tissue, biological fluid or organism. A proteomic sample may comprise about 0.000001%, 0.000005%, 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% of the polypeptide sequence diversity or content of a cell, tissue, or organism. A proteomic sample may comprise at least about 0.000001%, 0.000005%, 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% of the polypeptide sequence diversity or content of a cell, tissue, or organism. Alternatively or additionally, a proteomic sample may comprise no more than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, 0.0001%, 0.00005%, 0.00001%, 0.000005%, 0.000001%, or less than about 0.000001% of the polypeptide sequence diversity or content of a cell, tissue, or organism.

[0080] Samples of the present disclosure may be derived from any of a variety of environments. Samples may be collected in natural, artificial, altered, engineered, or anthropogenic environments. Samples may comprise a gas phase, liquid phase, solid phase, or a combination thereof. Samples may be collected from the gas phase, liquid phase, solid phase, or a combination thereof. Exemplary samples collected from the gas phase may include directly captured particulates or aerosols (e.g., dust, viral particles or aerosols), particulates captured by air filtration, or swabs of surface-sedimented airborne particulates. Exemplary samples collected from the liquid phase may include water samples, blood or other bodily fluid samples, or liquid rinses of solid surfaces. Exemplary samples collected from the solid phase may include soil samples, rock samples, biopsy or tissue samples, textile samples, or virtually any other solid material or substance.

[0081] A sample may be collected in any of a variety of environments. A sample may be collected by a human collector. A sample may be collected by an automated or robotic system. A sample may be collected with the aid of a collection device. A collection device may be a passive device (e.g., a filter or trap) or an active device (e.g., an intravenous or finger prick blood draw). A sample may be collected for any of a variety of purposes, including biological, ecological, paleontological, archeological, medical, diagnostic, clinical, or forensic purposes. A sample may be collected from a pre-existing sample, collected previously for a similar purpose (polypeptide analysis) or for a different purpose (nucleic acid analysis, phenotypic analysis, or an unanalyzed archival sample). A sample may be collected from any of a variety of locations, including air, water, soil, sand, rock, vegetation, wildlife, extraterrestrial, and anthropogenic objects. A sample may be collected in any of a variety of ecosystems within any known biome, including an oceanic, polar ice, arctic tundra, taiga, mountain, temperate deciduous forest, temperate evergreen forest, warm evergreen forest, tropical forest, tropical evergreen forest, chaparral, desert, savanna, semiarid, or temperate grassland ecosystem. A medical, clinical, or diagnostic sample may be collected in a medical, clinical, or diagnostic facility (e.g., hospital, medical lab, diagnostic facility), or in a non-medical environment (e.g., a farm or home). A medical, clinical, or diagnostic sample may be collected by a trained collector (e.g., a phlebotomist) or may be self-collected by a test subject (e.g., a urine or stool sample).

[0082] Standard polypeptides can be utilized during one or more of the sample collection processes set forth above or elsewhere herein. The standard polypeptides can be useful for characterizing the quality or quantity of the sample collected, characterizing the quality or quantity of sample polypeptides derived from the sample, characterizing a property of sample or the polypeptides obtained from the sample, or the like. For example, modification or degradation that is observed for sample polypeptides but not for standard polypeptides following collection and analysis can be attributed to the sample as opposed to the collection or analysis processes. Desired standard polypeptides can be introduced to a sample or fraction thereof prior to or during one or more of the sample collection processes set forth above or during another a process or step set forth above or elsewhere herein.

[0083] A sample may be collected into a sample vessel. A sample vessel may comprise any container capable of holding a sample. A sample vessel may comprise a vial, tube, jar, flask, cylinder, one or more wells of a multiwell plate, flow cell, capillary tube, vesicle, droplet, dipstick or bag. A sample vessel may be waterproof, corrosion-proof, leakproof, optically opaque or transparent, inert. A sample vessel may comprise a degradable material, such as a photolytic or thermally-decomposable polymer. A sample vessel may be sealable or unsealable. A sample vessel may be sterilized and / or sealed prior to the collection of a sample. A sample may be placed in a sample vessel in a sterile or non-sterile environment. A sample may be placed in a sample vessel by a method that prevents or minimizes contamination, e.g., glove bag, fume hood, positive pressure, gas flushing. A sample vessel may be used for transportation and / or storage of a sample to a location for a characterization assay. A sample vessel can contain a standard polypeptide before or after a sample is added to the vessel.

[0084] A sample may have a known or unknown age. A sample may have a known or unknown history of environmental insults. A sample may comprise a forensic, paleontological or archeological specimen. A sample may be collected immediately before a characterization assay. A sample may be collected from a living, senescent, deceased, or non-biological subject. In some cases, the age or history of a sample may be determined by a characterization assay. A sample may be fully or partially degraded, or fully intact. A standard polypeptide can be added to the sample to provide a basis for distinguishing degradation or modification that has occurred due to age or history of the sample from degradation or modification that occurs while preparing or analyzing the sample. More specifically, degradation or modification that occurs for a standard polypeptide(s) can be attributed to a technical artifact of the sample preparation or analysis process, whereas degradation or modification of sample polypeptides that are not also observed for the standard polypeptide(s) can be attributed to age or history of the sample. Comparison between sample polypeptides from samples having a different age or history can also be helpful in this regard.

[0085] A sample may be homogeneous or heterogeneous. A sample may be processed before a characterization assay to render the sample homogeneous. A sample may be homogenized by any appropriate method, including crushing, pulverization, grinding, mixing, and blending. A heterogeneous sample may be homogenized to produce a homogeneous sample. A sample may be subdivided or fractionated. A sample may be subdivided or fractionated to isolate, separate, refine, or concentrate constituent components of the sample. For example, a homogeneous tissue sample may be lysed to isolate membrane, cytoplasmic, and / or nuclear fractions. Whether or not a sample has been lysed, the contents of the sample can be fractionated by methods known in the art of biochemistry or analytical chemistry such as centrifugation, density-gradient centrifugation, chromatography (e.g. size exclusion chromatography, ionic exchange chromatography, reverse phase chromatography, affinity chromatography, thin layer chromatography etc.), liquid-phase extraction, solid-phase extraction, gel electrophoresis, filtration, distillation or the like. A homogeneous sample may be subdivided or fractionated to produce two or more heterogeneous fractions. A homogeneous sample may be subdivided or fractionated to produce at least one homogenous fraction, or to produce two or more homogeneous fractions, such as for performing replicate or repeat characterization assays.

[0086] Standard polypeptides can be utilized during one or more of the homogenization or fractionation processes set forth above or elsewhere herein. The standard polypeptides can be useful for characterizing a property of the process, characterizing a property of sample polypeptides obtained from the process and / or quantifying sample polypeptides obtained from the process. For example, standard polypeptide(s) having expected separation properties when subjected to one or more of the separation techniques set forth above can be used to identify properties of sample polypeptides that co-fractionate with the standard polypeptide(s). Exemplary properties include, but are not limited to, polypeptide length, polypeptide isoelectric point, polypeptide hydrophobicity, polypeptide hydrophilicity, polypeptide hydrodynamic radius, polypeptide charge, polypeptide mass, polypeptide charge to mass ratio, polypeptide pKa, presence or absence of post-translational modifications on polypeptides, polypeptide structure, stability of polypeptides to proteases, or the like. Desired standard polypeptides can be introduced to a sample or fraction thereof prior to or during one or more of the homogenization or fractionation processes set forth above or elsewhere herein.

[0087] A sample may have a known or unknown stability. Stability may refer to the tendency of the sample, or particular components within the sample, to undergo chemical changes that alter the composition of the sample relative to the composition at the moment of its collection. In some cases, stability may specifically refer to the tendency of particular components of the sample, such as a polypeptide fraction or sample polypeptide, to degrade after sample collection. A sample may be stable, conditionally stable, partially unstable, or unstable. A stable sample may experience little or no observable chemical degradation or alteration. A conditionally stable sample may experience little or no observable chemical degradation or alteration within a specified set of environmental conditions (e.g., within 24 hours when stored at 20 °C or less). For example, less than 10%, 5%, 1% or even less of a stable or partially stable sample may experience observable chemical degradation or alteration. A partially unstable sample may experience chemical degradation or alteration of particular components while other components remain unchanged. For example, less than 50%, 25%, 10%, 5%, 1% or even fewer of the polypeptide species in the sample may experience observable chemical degradation or alteration. An unstable sample may experience chemical degradation or alteration regardless of environmental conditions after sample collection. Global or component-specific instability or degradation may be described by a kinetic rate law, such as a first- or second-order reaction rate.

[0088] A sample may be treated before, during or after collection to prevent or inhibit degradation or alteration of a polypeptide fraction. The sample may be in any of a variety of states including, for example, an intact biological sample, crude lysate, fraction or other state set forth herein or known in the art. A sample may be treated to prevent or inhibit physical, chemical or biological processes that degrade or alter a polypeptide fraction or sample polypeptide. Polypeptides in a sample may be subject to chemical and / or biological processes such as oxidation, reduction, cleavage (hydrolytic or enzymatic), complexation, agglomeration, or post-translational modification. A sample containing one or more sample polypeptides may be treated to prevent or inhibit polypeptide degradation or alteration processes to obtain an instantaneous or near-instantaneous polypeptide or polypeptide panel at the time of sample collection. A sample may be treated to prevent or inhibit one or more specific chemical or biological processes that degrade or alter the sample polypeptide(s). For example, a protease inhibitor may be added to a sample to reduce or eliminate enzymatic polypeptide cleavage. A sample may be treated to cease all polypeptide activity, thereby preventing any biological or enzymatic polypeptide degradation or alteration processes. For example, a sample may be treated with a denaturant (e.g., guanidinium chloride or heat) to denature all polypeptides, thereby inhibiting or preventing polypeptide-mediated or polypeptide-related biological processes. A sample may be treated with one or more chemical agents to control the sample chemistry. A sample may be treated with one or more chemical agents, including acids, bases, buffers, salts, oxidizers, reductants, chelating agents, oxygen scavengers, surfactants, detergents, crosslinking reagents, aqueous solvents, and non-aqueous solvents.

[0089] Standard polypeptides can be useful for evaluating the stability of sample polypeptides during a treatment or process set forth above or elsewhere herein. For example, standard polypeptide(s) having known or expected susceptibility to various types of proteases or other reagents can be used during the treatment or process. The quantity or characteristic(s) of degradation that occurs for sample polypeptides can be determined by normalization with respect to the quantity or characteristic(s) of degradation that occurs for the standard polypeptides. Exemplary characteristics include, but are not limited to, presence or absence of protease recognition sites in a sample polypeptide, location of protease recognition sites in a sample polypeptide, and presence or absence of post-translational modifications that alter the extent of proteolysis. Desired standard polypeptides can be introduced to a sample or fraction thereof prior to or during one or more of the treatments or processes set forth above or elsewhere herein.

[0090] A sample may undergo one or more treatments that stop biological activities that affect polypeptide expression, composition, and / or abundance. The effect of such treatments may be to capture a near instantaneous profile of a proteome within a biological system. The one or more treatments may include stopping or inhibiting transcription and / or translation, degrading or removing nucleic acids, stopping or inhibiting post-translational modification of polypeptides, and stopping or inhibiting polypeptide degradation. A sample may undergo a treatment method such as heat treatment, cold treatment, freezing, enzymatic treatment (e.g., endonucleases, exonucleases, proteases), chemical treatment, or combinations thereof. Chemical treatments to stop or inhibit biological processes in samples may include addition of protease inhibitors, denaturants, acids, bases, salts, crosslinking reagents or surfactants. In some cases, chemical treatments may be combined with temperature treatments (e.g., heating, cooling) to increase the effectiveness of the treatment. A sample may undergo a treatment to stop or inhibit biological activity in the presence of a detergent. Without wishing to be bound by theory, the presence of a detergent during a sample treatment may separate some unwanted impurities from the polypeptide fraction of the sample, thereby enhancing or improving the separation and / or isolation of a polypeptide fraction from the sample. A treatment to stop biological activities may occur immediately after sample collection, or within a reasonable timeframe, for example no more than about 30 seconds (s), 1 minute (min), 2 mins, 3 mins, 4 mins, 5 mins, 10 mins, 15 mins, 20 mins, 30 mins, 45 mins, 1 hour (hr), 3 hrs, 6 hrs, 12 hrs, or no more than about 24 hrs. A treatment to stop biological activities may commence upon cell lysis, for example, by lysing the cell(s) in the presence of the treatment. Alternatively, a treatment to stop biological activities can be initiated after cell lysis, for example, within the time periods exemplified above or during a collection or fractionation process set forth herein. Polypeptide standards can be used to distinguish effects of the treatments on one or more sample polypeptides. For example, modification or degradation that is observed for sample polypeptides that are subjected to a treatment set forth above or elsewhere herein, but not observed for standard polypeptides in an untreated sample, can be attributed to the treatment.

[0091] A sample or sample polypeptide may be stored after collection. A sample or sample polypeptide may be stored after collection for a specified amount of time. A sample or sample polypeptide may be stored after collection at a specified temperature and / or pressure. A sample or sample polypeptide may be stored after collection at a specific humidity or under a specific inert atmosphere (e.g. in a vacuum or under inert gas). A sample or sample polypeptide may be stored after collection for a sufficient amount of time to permit the continuance or completion of a biological process including, for example, a biological process that was initiated prior to the collection process. A sample or sample polypeptide may be stored after collection for a sufficient amount of time to permit the completion of a post-collection treatment process such as a biological, chemical or physical process. The post-collection treatment process can involve only components that were present in the sample, or with the sample polypeptide, prior to initiating the collection process or alternatively the post-collection treatment process can involve one or more exogenous reagent that was added to the sample or sample polypeptide during or after initiation of the collection process. In some cases, a sample or sample polypeptide may be stored while awaiting or undergoing transport to a different location (e.g., from a collection site to an analysis facility).

[0092] A sample or sample polypeptide may be stored at a specified temperature. A sample or sample polypeptide may be stored at a temperature sufficient to freeze liquid in the sample. A sample or sample polypeptide may be stored at a temperature sufficient to prevent freezing of liquid in the sample. A sample or sample polypeptide may be stored at a temperature of about - 80 °C, - 70 °C, - 60 °C, - 50 °C, - 40 °C, - 30 °C, - 20 °C, - 10 °C, - 5 °C, 0 °C, 4 °C, 10°C, 20 °C, 30°C, 37 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, or about 95 °C. A sample or sample polypeptide may be stored at a temperature of at least about - 80 °C, - 70 °C, - 60 °C, - 50 °C, - 40 °C, - 30 °C, - 20 °C, - 10 °C, - 5 °C, 0 °C, 4 °C, 10 °C, 20°C, 30°C, 37 °C, 40 °C, 50 °C, 60°C, 70 °C, 80 °C, 90 °C, or at least about 95 °C. Alternatively or additionally, a sample or sample polypeptide may be stored at a temperature of no more than about 95 °C, 90 °C, 80 °C, 70 °C, 60 °C, 50 °C, 40 °C, 37 °C, 30 °C, 20 °C, 10 °C, 4 °C, 0 °C, - 5 °C, - 10 °C, - 20 °C, - 30 °C, - 40 °C, - 50 °C, -60 °C, - 70 °C, or about - 80 °C.

[0093] A sample or sample polypeptide may be stored for a specific amount of time. A sample or sample polypeptide may be stored at a specific temperature for a specific amount of time. A sample or sample polypeptide may be stored for no more than a specific amount of time to prevent alteration, degradation or aging of the sample or sample polypeptide. A sample or sample polypeptide may be stored for about 1 min, 30 mins, 1 hr, 3 hrs, 6 hrs, 12 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks (wks), 3 wks, 4 wks, 1 month (mth), 2 mths, 3 mths, 6 mths, 9 mths, 1 year (yr), 2 yrs, 3 yrs, 4 yrs, 5 yrs, 10 yrs, 20 yrs, 25 yrs, 50 yrs, or about 100 yrs. A sample or sample polypeptide may be stored for at least about 1 min, 30 mins, 1 hr, 3 hrs, 6 hrs, 12 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 wks, 3 wks, 4 wks, 1 mth, 2 mths, 3 mths, 6 mths, 9 mths, 1 yr, 2 yrs, 3 yrs, 4 yrs, 5 yrs, 10 yrs, 20 yrs, 25 yrs, 50 yrs, or at least about 100 yrs. Alternatively or additionally, a sample or sample polypeptide may be stored for no more than about 100 yrs, 50 yrs, 25 yrs, 20 yrs, 10 yrs, 5 yrs, 4 yrs, 3 yrs, 2 yrs, 1 yr, 9 mths, 6 mths, 3 mths, 2 mths, 1 mth, 4 wks, 3 wks, 2 wks, 10 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hrs, 6 hrs, 3 hrs, 1 hr, 30 mins, 1 min, or less than about 1 min.

[0094] A sample or sample polypeptide may be transported from a collection site to another site for subsequent processing or analysis. The sites may be within a single facility or campus. Alternatively, the sites may be located at different facilities or campuses, for example, in different cities, counties, states, countries, territories, time zones, or other geographically or politically delineated locations. During transport, a sample or sample polypeptide may be stored in a controlled environment. A controlled environment may prevent exposure of a sample to variable or detrimental environmental conditions. A controlled environment may prevent exposure of a sample to undesirable levels of heat, cold, humidity, radiation, vibration, pressure, or contaminants. A controlled environment may prevent a sample from undergoing biological, physical or chemical processes that affect the chemical composition or function of the sample. A controlled environment may prevent degradation or alteration of a sample polypeptide or polypeptide fraction of a sample. A sample or sample polypeptide may be stored in a controlled environment based upon instructions provided by an individual or entity providing the sample or sample polypeptide, or by an individual or entity receiving the sample or sample polypeptide. A controlled environment may include various forms of environmental regulation, including heaters, ovens, humidifiers, dehumidifiers, inert gas, vacuum, insulation, padding, shielding, lighting, and filtration (liquid or gas). A sample or sample polypeptide may be collected according to a prescribed or pre-defined protocol. A prescribed or pre-defined protocol may be specified by an individual or entity collecting the sample or sample polypeptide, an individual or entity that will receive the sample or sample polypeptide, or by an accepted standard protocol, such as an academic standard protocol, a commercial standard protocol, or an industrial standard protocol. A sample or sample polypeptide may be collected in a duplicate or replicate fashion. A sample may be divided into multiple sub-samples for the purposes of quality control or data verification. A sample may be divided into multiple sub-samples, with each sub-sample intended for a different type of disposal, storage, modification, manipulation or analysis. For example, a tissue sample may be divided into three separate sub-samples, with each sub-sample intended for either 1) a polypeptide assay, 2) nucleic acid characterization assay, or 3) a small-molecule metabolite characterization assay.

[0095] Standard polypeptides can be useful for evaluating the stability of sample polypeptides during a storage or transport process set forth above or elsewhere herein. For example, standard polypeptide(s) having known or expected susceptibility to various physical, chemical or biological processes that are known os suspected to occur during storage or transport can be used. The quantity or characteristic(s) of degradation or other change that occurs for sample polypeptides can be determined by normalization with respect to the quantity or characteristic(s) of degradation or other change that occurs for the standard polypeptides. Desired standard polypeptides can be introduced to a sample or fraction thereof prior to or during one or more of the storage or transport processes set forth above or elsewhere herein.

[0096] A sample or fraction thereof may be collected utilizing a collection kit. A collection kit may be specific to a particular assay to be performed on the sample. For example, a collection kit for a polypeptide assay may include polypeptide-specific reagents to protect and / or preserve polypeptides within a sample. A collection kit may include one or more sample vessels, one or more reagents, instructions for use of the sample collection kit and optionally intermediate sample vessels, a sealant for the vessel(s), a label for the vessel(s) such as a barcode or radio frequency identification device (RFID), or packaging for transport and / or storage of the sample vessel(s). A sample collection kit may include one or more reagents for any of a variety of purposes, including sample preservation, sample stability, sample quality control, processing and / or purification, and sample storage. A sample collection kit may include reagents such as buffers, acids, bases, solvents, denaturants, surfactants, detergents, reactants, labels (e.g., fluorophores, radiolabels), indicator dyes, enzymes, enzyme inhibitors, oxygen scavengers, water scavengers, humectants, affinity reagents (e.g., antibodies), or other capture agents (e.g., biotinylated particles). A sample collection kit may include one or more reagents in liquid or solid form. A sample collection kit may include one or more separate reagents and / or polypeptide standards that are added to the sample collection vessel before or after sample collection. A sample collection kit may include one or more reagents and / or polypeptide standards that are provided within the sample collection vessel. For example, reagents and / or polypeptide standards may be provided in a crystallized or coated form on a surface of the collection vessel, or may be in a liquid solution within the collection vessel. A sample collection kit may have a pre-determined shelf life due to the provided reagents or other materials.

[0097] A sample collection kit for a polypeptide assay may vary depending upon the type of characterization to be performed. For example, a collection kit may be specific to a sample component (e.g., blood plasma from whole blood) or may be non-specific to a sample (e.g., full proteome from a tissue or blood sample). A sample collection kit may be sample-specific (e.g., blood, urine, tissue, fecal, cell swab, etc.) or may be sample-agnostic. A sample collection kit may include one or more polypeptide standards that are useful for characterizing a process carried out with the kit or product produced by the process. A sample-agnostic collection kit may be capable of preserving and / or preparing a protein fraction for analysis regardless of the nature or form of the sample.

[0098] A sample collection kit for a polypeptide assay may be utilized according to a provided set of instructions. The instructions may be directed to use of polypeptide standards in accordance with teachings set forth herein. A sample collection kit may be utilized by a technician or self-collecting subject. A technician utilizing a sample collection kit may be specifically trained in the proper utilization of the sample collection kit. A sample collection kit protocol may employ one or more intermediate steps before collection of the sample is complete. Intermediate steps during sample collection may be performed in the sample collection vessel or in a separate medium (provided with the kit or provided by the collector). For example, a blood sample may be fractionated by a phlebotomist, with only the red blood cell or plasma fraction saved for collection. A sample collection kit may include indicator dyes, litmus strips, or other methods of confirming successful sample collection and / or preparation. A sample collection kit may include a sealant (e.g., an adhesive or sticker) to ensure that a sample has not been tampered with or damaged during storage or transport. A sample collection kit may include a label for sample tracking by the collector or the analysis facility. A label for a sample collection vessel may include a serial number, RFID, bar code or QR code. A label for a sample collection vessel may be pre-printed or pre-applied to a sample collection vessel, or may be placed by a collector.

[0099] A sample kit may comprise one or more reagents that are intended to perform one or more steps of a sample preparation process. For example, a sample kit may comprise one or more reagents that lyse cells, stop biological processes (e.g., protein synthesis or degradation), stop degradative processes (e.g., oxidation), functionalize sample polypeptides to add reactive handles to the polypeptides, crosslink polypeptides, or couple sample polypeptides to anchoring groups to form polypeptide composites. In some cases, a sample kit may comprise two or more reagents that are added in a step-wise fashion according to a set of instructions. In other cases, a sample kit may comprise one or more reagents that are added simultaneously to perform one or more sample preparation processes set forth herein. A sample kit can include one or more standard polypeptides that are added in a stepwise fashion or simultaneously to perform one or more sample preparation processes set forth herein. In some configurations, the use of a sample kit may comprise one or more of the steps of: 1) adding a sample to a handling or storage vessel; 2) adding one or more reagents or standard polypeptide to the handling or storage vessel; 3) combining the sample, the one or more reagents and / or the standard polypeptide(s) in the handling or storage vessel to form a sample mixture; 4) forming a plurality of sample polypeptide composites and / or standard polypeptide composites in the handling or storage vessel in the sample mixture; 5) applying the sample mixture to a solid support; and 6) removing a waste fraction from the solid support to form a polypeptide array.

[0100] A sample may undergo one or more sample pretreatment processes during or after separation from a natural source. A sample pretreatment process may occur before the sample undergoes a sample preparation process for a polypeptide assay. In particular configurations, a sample pretreatment process may occur before, during or after a step of a sample preparation process set forth herein. A sample pretreatment process may comprise one or more in vitro processes that alter a sample or a fraction of polypeptides within the sample. A sample pretreatment process may alter a sample or a fraction of polypeptides within the sample in a predictable fashion (e.g., treatment with an enzyme with a known and / or characterized selectivity or reactivity). A sample pretreatment process may alter a sample or a fraction of polypeptides within the sample in an unknown, unpredictable, and / or uncharacterized fashion. For example, a sample may be treated with a drug or drug analog that has the possibility of physically or chemically altering one or more polypeptides within the sample. Exemplary sample pretreatment processes may include, without limitation, oxidation, reduction, denaturation, lysis, homogenization, digestion, labeling (e.g., fluorophores, barcodes, radiolabels, isotopes), dilution, concentration, disruption of polypeptide complexes, formation of polypeptide complexes, crosslinking of polypeptides to other biomolecules, addition of post-translational modifications, and removal of post-translational modifications. In some cases, a sample pretreatment process may comprise the cross-linking of a polypeptide to one or more neighboring or adjacent biomolecules (e.g., polypeptides, nucleic acids, polysaccharides, lipids, etc.). Cross-linking of polypeptides to neighboring biomolecules may capture short-term or long-term spatial associations between related biological components that may be disrupted by subsequent sample preparation processes.

[0101] A sample pretreatment process may include contacting a sample, or fraction thereof, with an enzyme that causes post-translational modification of a polypeptide (e.g. sample polypeptide or standard polypeptide). An enzyme that causes any of a variety of post translational modifications can be used including, but not limited to, enzymes that add or remove a moiety set forth herein. For example, a sample, or fraction thereof, can be treated with a kinase to add a phosphate moiety to a polypeptide or with a phosphatase that removes a phosphate moiety from a polypeptide. The phosphate moiety can be present on a serine, threonine, tyrosine, aspartate or glutamate residue of the polypeptide. A sample, or fraction thereof, can be treated with an N-terminal acetyltransferase to add an acetyl moiety to the N-terminus of a polypeptide or with an acetyltransferase that adds an acetyl moiety to a lysine of the polypeptide. Acetyl moieties can optionally be removed by a deacetylase enzyme. A glycosyltransferase or galactosyltransferase can be used to add a carbohydrate moiety to a polypeptide. For example, a serine or threonine residue can have an O-linked glycosyl moiety, or an asparagine residue can have an N-linked glycosyl moiety. All or part of a carbohydrate moiety can be removed from a polypeptide using an appropriate enzyme such as α2-3,6,8,9-neuraminidase; β1,4-galactosidase; β-N-acetylglucosaminidase; endo-α-N-acetylgalactosaminidase or PNGase F. A proline, lysine, asparagine, aspartate or histidine amino acid can be hydroxylated by a hydroxylase. A polypeptide can be methylated by an arginine methyltransferase or lysine methyltransferase. A polypeptide can be ubiquitinated using ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin ligase. Ubiquitin moieties can be removed form polypeptides using a deubiquitinating enzyme or protease.

[0102] A sample pretreatment process may include contacting a sample, or fraction thereof, with chemical reagent that produces a chemically modified polypeptide (such as a chemically modified sample polypeptide or a chemically modified standard polypeptide). Cysteine can be reacted with 5,5-dithio-bis-(2 nitrobenzoic acid) (DTNB) to form TNB modified cysteine. Optionally, the TNB modified cysteine can be reacted with C≡N -< , SO 3 2-< , or RS -< nucleophiles to form cysteine-S-CN, cysteine-S-SO 3 or cysteine-S-S-R adducts, respectively, wherein R is an organic moiety such as a hydrocarbon. Cysteine can also be modified with an alkylating reagent (e.g. amide, ketone, carboxylic acid, ester or heterocyclic strained ring) or alkene (e.g. 4-vinylpyridine or maleimide) to form a cysteine thioether. Amino moieties in polypeptides can be acylated, for example, via reaction with succinic anhydride or maleic anhydride; guanidinated, for example, via reaction with O-methylisourea; amidinated, for example, by reaction with methylacetimidate or methylpicolinimidate; acetylated, for example, via reaction with acetic anhydride; carbamylated, for example, via reaction with cyanate; arylated, for example, via reaction with 1-fluoro-2,4-dinitro-benzene or trinitrobenzenesulfonic acid; deaminated, for example, via reaction with nitrous acid; acylated, for example, via reaction with succinic anhydride; or reductively alkylated, for example, via reaction with formaldehyde and NaBH 4 . Carbonyl moieties in polypeptides can be converted to esters or amides using reagents known in the art. Particularly useful reagents for modifying carbonyl moieties are carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or dicyclohexylcarbodiimide. Histidine can be reacted with ethoxy formic anhydride or subjected to Bamberger cleavage. Photoreactive reagents can be useful for modifying a variety of moieties in a polypeptide. Exemplary reagents and techniques for chemical modification of polypeptides are set forth, for example, in Means and Feeney Bioconjugate Chem. 1, 2-12 (1990); Matthews et al. Meth. Enz. 208:468-496 (1991); and Glazer, The Proteins 3rd Ed. Vol. 2 (1976).

[0103] A sample pretreatment process may include crosslinking a polypeptide to another reagent or entity, such as a polypeptide. Exemplary crosslinkers include heterobifunctional or homobifunctional crosslinkers, both of which have a spacer moiety flanked by reactive moieties. The spacers can either be cleavable, for example, using conditions that are relatively mild to preserving the integrity of the crosslinked species, or the spacers can be effectively non-cleavable under the conditions of use. Zerolength crosslinkers, having no spacer moiety, can also be useful. Any of a variety of reactive moieties including those exemplified above or others known in the art can be used. Exemplary crosslinking reagents are set forth in Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, Boca Raton, FL (1991) or available commercially from Sigma Aldrich (St. Louis, MO) or Thermo Fisher (Waltham, MA).

[0104] A standard molecule (e.g. standard polypeptide) can be utilized during one or more of the sample pretreatment processes set forth above or elsewhere herein. The standard can be useful for characterizing the reactivity of reagents used for modifying polypeptides. For example, a standard polypeptide having known reactivity toward a chemical reagent or enzyme can be used to measure the extent to which the reactions occurred. Another useful type of standard is a standard polypeptide or other molecule that is modified when an unwanted side reaction occurs, such that the modification can be observed to determine if unwanted reactions occurred for one or more polypeptide in a sample. Pretreatment standard polypeptides (or other pretreatment standard molecules) can be introduced to a sample or fraction thereof prior to or during a pretreatment process or step set forth above or elsewhere herein.

[0105] A sample may comprise a plurality of sample polypeptides. In some cases, a sample preparation method may be configured to capture or collect all or a maximal quantity of sample polypeptides from a sample. In other cases, a sample preparation may be configured to capture or collect a specific subset of sample polypeptides from a sample. A sample may comprise sample polypeptides and non-target polypeptides, where the non-target polypeptides are intended to be excluded after collection or capture of the sample polypeptides. Non-target polypeptides may include damaged polypeptides, degraded polypeptides, truncated polypeptides, small polypeptides (e.g., less than about 100, 75, 50, 40, 30, 25, 20, 15, 10, or less than 10 amino acids) or large polypeptides (e.g., greater than about 5000, 10000, 15000, 20000, 25000, or more than 25000 amino acids). A sample polypeptide content before a sample preparation method may be measured, for example by UV absorption, Biuret methods, colorimetric dye methods, or fluorescent dye methods. A sample polypeptide content may be measured on a mass or molar basis. The same measurements can be used for standard polypeptides. A sample preparation method may produce a polypeptide fraction or polypeptide array comprising a plurality of sample polypeptides. The plurality of sample polypeptides may comprise at least about 0.000001%, 0.000005%, 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more than 99.99999% of the sample polypeptide content of a sample on a mass or molar basis. Alternatively or additionally, the plurality of sample polypeptides may comprise no more than about 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, 0.0001%, 0.00005%, 0.00001%, 0.000005%, 0.000001%, or less than 0.000001% of the sample polypeptide content of a sample on a mass or molar basis.

[0106] A plurality of polypeptides can be characterized on the basis of polypeptide sequence content. The polypeptide sequence content for a fraction of polypeptides obtained from a particular sample can be measured as the percent of sequences in the sample that are present in the fraction. A basis for polypeptide sequence content may include genetic analysis (e.g., known or predicted translated amino acid sequences of identified genes) and / or measurements of polypeptide sequence content in other polypeptide samples. The measure is typically grounded in a sequence string length (also referred to as a "word' length) of a specified length, for example, a string length of 3 amino acids, 5 amino acids, 8 amino acids, 10 amino acids, 12 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 50 amino acids, 100 amino acids or more. Accordingly, the number of sequences having a particular string length in a sample is compared to the number of sequences having that string length in a fraction obtained from the sample. The sample can be a biological source or sample derived from a biological source. The fraction can be a plurality of polypeptides in a liquid sample, in an array or in another composition set forth herein. The fraction of sample polypeptides may comprise a polypeptide sequence content that includes at least about 0.000001%, 0.000005%, 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more than 99.99999% of the polypeptide sequence content of a sample, when comparing sequences having a selected sequence string length. Alternatively or additionally, the fraction of sample polypeptides may comprise a polypeptide sequence content that includes no more than about 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, 0.0001%, 0.00005%, 0.00001%, 0.000005%, 0.000001%, or less than 0.000001% of the polypeptide sequence content of a sample, when comparing sequences having a selected sequence string length.

[0107] An internal standard may be combined with a sample before, during, or after sample collection. The internal standard may comprise polypeptides or other components that permit quality control of any subsequent sample processing. Internal standards may be used to assess the quality and / or viability of a sample after handling, storage, or sample preparation processing. Internal standards may be used to determine the success or efficiency of sample preparation processing. Internal standards may permit normalization within a sample or across a group of samples. For example, an internal standard may be utilized to determine the differential separation, functionalization, or conjugation of particular polypeptides within a sample on the basis of a particular property (e.g., size, hydrophobicity, surface charge density, etc.). In another example, an internal standard may be utilized across a group of samples to assess the variability of a sample preparation process based upon observed differences in presence of the internal standards in each prepared sample.

[0108] An internal standard comprising a plurality of standard polypeptides may be combined with a plurality of sample polypeptides during a sample preparation method. The total quantity of a standard polypeptides combined with a plurality of sample polypeptides may be adjusted depending upon the intended use of the standard polypeptides for quantitative or qualitative characterization of a sample preparation method. A mass fraction of sample polypeptides in a polypeptide mixture may be calculated as a ratio or percentage of a plurality of sample polypeptides to the total quantity of the plurality of sample polypeptides and the total quantity of a plurality of standard polypeptides (e.g., sample collection standard polypeptides, sample handling standard polypeptides, separation standard polypeptides, functionalization standard polypeptides, coupling standard polypeptides, and attachment standard polypeptides) provided to a sample preparation method. For example, a mixture of a plurality of sample polypeptides with a mass of 9 nanograms and a plurality of separation standard polypeptides with a mass of 1 nanograms would have a sample polypeptide mass fraction of 90% or 0.9. A sample polypeptide mixture comprising a plurality of standard polypeptides may have a sample polypeptide mass or molar fraction of at least about 0.000001%, 0.000005%, 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or more than 99.99999%. Alternatively or additionally, a sample polypeptide mixture comprising a plurality of standard polypeptides may have a sample polypeptide mass or molar fraction of no more than about 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, 0.0001%, 0.00005%, 0.00001%, 0.000005%, 0.000001%, or less than 0.000001%.

[0109] A sample polypeptide mixture may comprise a plurality of standard polypeptides, in which the plurality of standard polypeptides comprises one or more types of standard polypeptides (e.g., coupling standards, separation standards, attachment standards, etc.), such as, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ,16, 17, 18, 19, 20, or more than 20 types of standard polypeptides. Alternatively or additionally, a sample polypeptide mixture may comprise a plurality of standard polypeptides, in which the plurality of standard polypeptides comprises one or more types of standard polypeptides, such as, for example, no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 , or less than 2 types of standard polypeptides. In some cases, differing types of standard polypeptides may be present in a sample polypeptide mixture in identical or similar amounts (e.g., within about 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less than 0.1% of an average amount or concentration). Differing types of standard polypeptides may be present in differing amounts in a sample polypeptide mixture. For example, a plurality of standard polypeptides may contain about 20% coupling standard polypeptides and 10% separation standards on a molar basis. Differences in amount of a type of standard may arise due to sample preparation conditions. For example, standards may be introduced in similar or identical quantities at each step of a sample prepration process, in which differences in amounts of each type of standard may be correlated to a relative efficiency of a step of the process. Alternatively, standards may be introduced in differing amoutns throughout a sample preparation process for other reasons, such as the relative detection sensititivity of a type of standard, or a relative expense of a particular type of standard. A plurality of standard polypeptides may comprise at least about 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more than 99.% of a type of standard polypeptide on a mass or molar basis. Alternatively or additionally, a plurality of standard polypeptides may comprise no more than about 99.9%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001%, 0.0000001%, 0.00000001%, 0.000000001%, or less than 0.000000001% of a type of standard polypeptide on a mass or molar basis.Internal Standards

[0110] A polypeptide fraction may be combined with one or more internal standards to form a polypeptide mixture. Internal standards may include standard polypeptides (also referred to herein as "internal standard polypeptides"), or internal standard non-polypeptides. Any of a variety of the standard polypeptides set forth herein can be used as internal standard polypeptides. Several configurations of the methods, compositions and systems of the present disclosure are exemplified with reference to standard polypeptides. It will be understood that internal standard non-polypeptides can be used as alternatives or supplements in the methods, compositions and systems of the present disclosure.

[0111] An internal standard may be any composition, such as a standard polypeptide, that is combined with a polypeptide fraction in order to: 1) provide a history of the polypeptide fraction any time during or after sample collection, 2) provide qualitative or quantitative measures of efficiency in various processing steps, 3) provide positive or negative controls for reagents or steps used in a polypeptide assay, or 4) a combination thereof. An internal standard may provide a history of a polypeptide fraction by displaying chemical properties that are linked to aspects of sample collection, handling, and storage. For example, an internal standard may provide information on sample age (e.g., by conversion of a solid support standard by a slow chemical reaction), sample temperature (e.g., polypeptides that aggregate at high or low temperatures), sample pH (e.g., indicator dyes), sample oxidation, or sample degradation.

[0112] An internal standard may be provided to a polypeptide fraction as a composition comprising one or more components. Internal standards may include internal standard polypeptides, and internal standard non-polypeptides. Internal standards may include small molecule components such as dyes, indicators, and enzymatic substrates. Internal standards may include biomolecule monomers (e.g., nucleotides, amino acids) with detectable labels (e.g., radiolabels, deuterium). Incorporation of labeled biomolecule monomers may provide evidence of intended or unintended on-going biological processes in a sample. In some cases, polypeptides containing incorporated labeled amino acids may be excluded from a polypeptide assay due to their likely formation or modification after sample collection. In other cases, polypeptides containing incorporated labeled amino acids may be included in a polypeptide assay due to their likely formation or modification after sample collection. In some cases, an internal standard may comprise a paired solid support and enzyme that provides an estimate of sample age or storage history. For example, an enzyme / substrate system may be included with a frozen sample to show improper storage if the presence of a converted substrate is detected. In another example, an enzyme / substrate system that produces a detectable product on a timescale comparable to the period of time that a sample is stored, transported or collected may be used to qualitatively or quantitatively characterize a sample after collection, transport or storage. Enzyme / substrate systems may be chosen based upon the expected length of collection, handling, and / or storage.

[0113] Internal standards may comprise a plurality of internal standard polypeptides. Internal standard polypeptides may be chosen based upon the information for which they are utilized to provide, the nature of the polypeptide fraction with which they will be combined, and the nature of the polypeptide assay within which they will be utilized. An internal standard, such as sample collection standard polypeptides, sample handling standard polypeptides, separation standard polypeptides, functionalization standard polypeptides, coupling standard polypeptides, and attachment standard polypeptides, comprising a plurality of polypeptides may comprise a single species of polypeptide. An internal standard, such as sample collection standard polypeptides, sample handling standard polypeptides, separation standard polypeptides, functionalization standard polypeptides, coupling standard polypeptides, and attachment standard polypeptides, comprising a plurality of polypeptides may comprise one or more species of polypeptide. An internal standard, such as sample collection standard polypeptides, sample handling standard polypeptides, separation standard polypeptides, functionalization standard polypeptides, coupling standard polypeptides, and attachment standard polypeptides, comprising a plurality of polypeptides may comprise two or more different species of polypeptide. An internal standard comprising a plurality of polypeptides may comprise a species of polypeptide that is a native species of a sample with which the internal standard polypeptides will be combined (e.g., an E. coli polypeptide as an internal standard for an E. coli sample). An internal standard comprising a plurality of polypeptides may not comprise any species of polypeptide that is a native species of a sample with which the internal standard polypeptides will be combined (e.g., no E. coli polypeptides contained in an internal standard for an E. coli sample). An internal standard may comprise polypeptides derived from the same source as a sample polypeptide. For example, an internal standard for a human sample may comprise polypeptides derived from the same human or a different human. Alternatively, an internal standard may comprise polypeptides derived from a differing source from a sample polypeptide. Internal standard polypeptides, such as sample collection standard polypeptides, sample handling standard polypeptides, separation standard polypeptides, functionalization standard polypeptides, coupling standard polypeptides, and attachment standard polypeptides, may be derived from a biological source, such as an animal, plant, bacteria, fungi, protist, archaea, or virus. An internal standard may comprise polypeptides derived from a biological source that is orthogonal to a sample with which the internal standard polypeptides will be combined. For example, a human sample may be combined with internal standard polypeptides from a species of bacteria or plant. An internal standard, such as sample collection standard polypeptides, sample handling standard polypeptides, separation standard polypeptides, functionalization standard polypeptides, coupling standard polypeptides, and attachment standard polypeptides, may comprise polypeptides derived from a curated source. An internal standard may comprise polypeptides that are synthetic or artificial polypeptides. An internal standard may comprise a polypeptide with a modified or non-natural amino acid (e.g., a fluorescent amino acid, a biotinylated amino acid). An internal standard may comprise an engineered polypeptide with a known or unknown biological function. An internal standard may comprise an engineered polypeptide with no known biological function. Engineered polypeptides may include polypeptides with amino acid sequences that are not known to exist in nature but are readily recognizable by a detection method (e.g., NAVTILVS, SAMPLEPREP, KEITHWILLIAMGNESHINPHDPATENTAGENT). An internal standard may comprise an isotopically labeled polypeptide where some atoms are replaced by isotopes to alter the molecular mass without altering other polypeptide properties. An internal standard may comprise a tag that is configured to make the internal standard readily identifiable or detectable during an assay. A tag may include a polypeptide tag (e.g., a peptide tag, a covalent peptide tag, a protein tag, etc.) or an oligonucleotide tag. A polypeptide array may comprise two or more differing types of internal standards (e.g., a functionalization standard and a purification standard), in which each type of standard is labeled with a differing tag that readily identifies the type of standard on the polypeptide array.

[0114] In some configurations, a sample may comprise one or more polypeptides or non-polypeptide compounds that may be utilized as internal standards. Depending upon sample types, certain polypeptide or non-polypeptide species may be present in a sample in a predictable fashion and may be present as impurities, inerts, or passengers during a polypeptide sample preparation process or a substituent step thereof. The presence or absence of the polypeptide or non-polypeptide species may be qualified or quantified after a polypeptide sample preparation process or a substituent process thereof to determine the outcome of the overall process or the substituent step.

[0115] A polypeptide standard may have a particular sequence length. A polypeptide standard may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000 or more than 30000 amino acid residues in length. Alternatively or additionally, a polypeptide standard may be no more than about 30000, 20000, 15000, 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 500, 450, 400, 350, 300, 250, 200, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less than 10 amino acids in length. A polypeptide standard may have a molecular weight of at least about 500 Daltons (Da), 1 kiloDalton (kDa), 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1 megaDalton (MDa), 2 MDa, 3 MDa or more than 3 MDa. Alternatively or additionally, a polypeptide standard may have a molecular weight of no more than about 3 MDa, 2 MDa, 1 MDa, 900 kDa, 800 kDa, 700 kDa, 600 kDa, 500 kDa, 400 kDa, 300 kDa, 200 kDa, 100 kDa, 90 kDa, 80 kDa, 70 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 20 kDa, 10 kDa, 5 kDa, 1 kDa, 500 Da, or less than 500 Da. An internal standard may comprise a plurality of polypeptides with a known length or weight distribution. For example, an internal standard may comprise a monomodal distribution of proteins from a weight of about 1 kDa to 20 MDa, with a peak near 500 kDa. Alternatively, an internal standard may comprise a polymodal distribution with groupings of polypeptides between 1 kDa to 50 kDa, 50 kDa to 250 kDa, 250 kDa to 1 MDa, and 1 MDa to 20 mDa.

[0116] Alternatively or additionally, internal standards may be composed with polypeptide distributions reflecting other polypeptide properties or characteristics. Internal standards may contain mixtures of polypeptides distinguished by properties such as hydrophobicity, hydrophilicity, isoelectric point, folded / misfolded, modification state (e.g., phosphorylated / unphosphorylated; inquinated / non-inquinated), pKa, mass, charge to mass ratio, presence or absence of post-translational modifications, solubility in aqueous solvents, membrane solubility, etc. Internal standards may be composed to approximate or imitate an expected property distribution for a polypeptide fraction or sample polypeptide derived from a sample. Internal standards may be composed to approximate or imitate an expected property distribution for a polypeptide fraction or sample polypeptide derived from a process set forth herein. Internal standards may be composed to deviate from an expected property distribution for a polypeptide fraction derived from a sample or process set forth herein. For a given polypeptide property or characteristic (e.g., size, weight, isoelectric point, etc.), a polypeptide distribution in an internal standard may follow any type of distribution, such as a Poisson distribution, binomial distribution, beta-binomial distribution, hypergeometric distribution, or bimodal distribution.

[0117] An internal standard may further comprise non-polypeptide components, such as small molecule compounds or non-polypeptide macromolecules. Non-polypeptide internal standard components may be selected as analogs or proxies for polypeptides or may be selected as representative of chemical and / or physical properties of polypeptides. For example, non-polypeptide components (e.g., polymeric nanoparticles) may be prepared to represent an expected range of one or more polypeptide properties, such as size, hydrodynamic radius, hydrophobicity, hydrophilicity, surface charge density, etc. Non-polypeptide internal standards may include chemical indicators (e.g., pH indicators, oxygen or radical scavengers) that provide measurable or detectable information on sample conditions. A non-polypeptide internal standard component may comprise an inert, undetectable, or orthogonal species that is not detected in a sample unless experiencing deleterious or detrimental environmental conditions (i.e., a "canary-in-the-coal-mine" compound). For example, an internal standard may comprise a branched or dendrimeric compound comprising cleavable fragments under certain conditions (e.g., high or low pH). The cleavable fragments may only be detected (e.g., by mass spectrometry, IR, UV, fluorescence, etc.) if released by cleavage of the compound. In another example, an internal standard may comprise a compound that is initially inert or undetectable but, under certain conditions, reacts to form a detectable compound (e.g., forming a fluorophore). The newly formed detectable compound may form a detectable species that is orthogonal to other detectable species utilized on a polypeptide assay platform (e.g., forming a fluorophore with an emission wavelength that is unique from other utilized fluorophores). A non-polypeptide internal standard may comprise a designed or engineered compound that provides multiple reporters for different conditions. An internal standard may comprise a branched or dendrimeric compound comprising polypeptides and non-polypeptide standards, thereby limiting the amount of required polypeptides or other compounds to be supplied in a polypeptide internal standard.

[0118] An internal standard may be added to a sample or a polypeptide fraction derived from a sample at any point prior to or during the preparation of a polypeptide fraction, including, for example, prior to or during sample collection, sample handling, sample storage, polypeptide separation, polypeptide purification, polypeptide composite formation, attachment of a polypeptide to a solid support and polypeptide composite deposition. A polypeptide fraction may comprise a plurality of internal standards, where each internal standard of the plurality of internal standards is added before or during a particular processing step, or is added for use of characterizing a particular processing step. As such, some internal standards may be referred to herein with reference to the processing step for which they are utilized. For example, a polypeptide assay may utilize sample collection internals standards, sample storage internal standards, sample separation internal standards, sample chromatography internal standards, sample extraction internal standards, sample functionalization internal standards, sample, coupling internal standards, and binding internal standards. In some cases, a polypeptide fraction may comprise a single internal standard that is added at a particular process during the preparation of a polypeptide fraction.

[0119] Prior to use as an internal standard, the behavior of an internal standard may be characterized for each known or anticipated processing step in a sample preparation method. The behavior characterization may include characterizations of component loss, component degradation, and process efficiency (e.g., percentage of polypeptides functionalized during a functionalization reaction). For internal standards comprising multiple types or species of polypeptides, the behavior characterizations may be evaluated as a function of the types or species of polypeptides. For example, percentage of internal standard polypeptides functionalized during a functionalization reaction may be provided as a function of polypeptide property (e.g., size, weight, isoelectric point, etc.). Process efficiencies for each known step of a polypeptide preparation process (e.g., separation, functionalization, coupling to anchoring groups, coupling to a solid support) may be characterized as a bulk property of an internal standard, or as a function of polypeptide distribution within an internal standard.

[0120] The behavior of an internal standard during a sample preparation method may be correlated to the behavior of a sample during the sample preparation method. In some cases, the behavior of an internal standard may be directly proportional to the behavior of a sample during a sample preparation method. For example, on a weight basis, the percentage of sample polypeptides recovered from a separation process may be proportional to the percentage of internal standard polypeptides recovered from the separation process. In another example, a known property of an internal standard can be useful in attributing a similar property to sample polypeptides that co-fractionate with the internal standard. In other cases, the behavior of an internal standard, such as sample collection internals standards, sample storage internal standards, sample separation internal standards, sample functionalization internal standards, sample, coupling internal standards, and binding internal standards, may be non-proportional to the behavior of a sample during a sample preparation method. For example, on a mass basis, the percentage of sample polypeptides recovered from a separation process may increase or decrease relative to the percentage of internal standard polypeptides recovered from the separation process.

[0121] In some cases, an overall efficiency of preparation for a polypeptide fraction derived from a sample preparation method may be calculated based upon efficiencies of individual steps as determined by the measurement of internal standards. An overall efficiency of preparation for a polypeptide for a polypeptide fraction during a sample preparation method may be calculated, for example, as a product of efficiencies for the N processing steps of the method. The overall efficiency may be expressed as a product of efficiencies for steps 1 to N, expressed as: E O = ∏ 1 N E n α where E O is the overall efficiency, E n is the efficiency of the nth step of the sample preparation method (e.g., collection, storage, separation of polypeptides, coupling of anchoring groups, coupling to solid support, etc.), and α is a proportionality exponent. When the proportionality exponent, α, has a value of 1, this indicates that the behavior of the sample in processing step n is proportional to the behavior of the internal standard during the processing step. When the proportionality exponent, α, has a value greater than 1, this indicates that processing step is less efficient for the sample than the internal standard. When the proportionality exponent, α, has a value less than 1, this indicates that processing step is more efficient for the sample than the internal standard. Equation 1 may be utilized to estimate changes in overall efficiency for sample preparation when processing methods are altered (e.g., adding steps, removing steps, changing processes, etc.).

[0122] An internal standard polypeptide, such as a separation standard polypeptide or a coupling standard polypeptide may comprise a detectable label. A detectable label may be a pendant group that is coupled or conjugated to an internal standard polypeptide (e.g., a fluorophore) or may be directly incorporated within the polypeptide (e.g., a radiolabel or atomic isotope). A detectable label coupled, conjugated, or incorporated with an internal standard polypeptide may comprise a fluorescent group, a luminescent group, a phosphorescent group, an enzyme, a radiolabel, a moiety comprising a non-standard isotope (e.g., heavy or light isotope), or a nucleic acid barcode. In some configurations, a detectable label may comprise a functional group comprising one or more non-standard isotopes that are readily detectable (e.g., by NMR or mass spectrometry). In some configurations, a detectable label may comprise an orthogonal functional group that would provide specific identification or analysis of an internal standard polypeptide. In some configurations, an internal standard polypeptide may comprise a fluorescent labeling group selected from the group consisting of FITC, Alexa Fluor ®< 350, Alexa Fluor ®< 405, Alexa Fluor ®< 488, Alexa Fluor ®< 532, Alexa Fluor ®< 546, Alexa Fluor ®< 555, Alexa Fluor ®< 568, Alexa Fluor ®< 594, Alexa Fluor ®< 647, Alexa Fluor ®< 680, Alexa Fluor ®< 750, Pacific Blue, Coumarin, BODIPY FL, Pacific Green, Oregon Green, Cy3, Cy5, Pacific Orange, TRITC, Texas Red, R-Phycoerythrin, and Allophycocyanin (APC). In some cases, the label may be an Atto dye, for example Atto 390, Atto 425, Atto 430, Atto 465, Atto 488, Atto 490, Atto 495, Atto 514, Atto 520, Atto 532, Atto 540, Atto 550, Atto 565, Atto 580, Atto 590, Atto 594, Atto 610, Atto 611, Atto 612, Atto 620, Atto 633, Atto 635, Atto 647, Atto 655, Atto 680, Atto 700, Atto 725, Atto 740, Atto MB2, Atto Oxa12, Atto Rho101, Atto Rho12, Atto Rho13, Atto Rho14, Atto Rho3B, Atto Rho6G, or Atto Thio12.

[0123] In some cases, the resulting composition of a sample preparation method may be a polypeptide array comprising a plurality of sample polypeptides and internal standard polypeptides coupled to a solid support with an overall efficiency of sample polypeptide capture that can be calculated based upon the internal standards. In some configurations, the composition may comprise a plurality of polypeptides, where the plurality of polypeptides comprises a plurality of sample polypeptides, a plurality of separation standard polypeptides, and a plurality of coupling standard polypeptides. The composition may further comprise a plurality of anchoring groups, where each polypeptide of the plurality of polypeptides may be coupled to an anchoring group of the plurality of anchoring groups to form a plurality of polypeptide composites, each of the polypeptide composites including a polypeptide from the plurality of polypeptides and an anchoring group from the plurality of anchoring groups. Further, the composition may comprise a solid support, where each anchoring group of the plurality of anchoring groups may be coupled to the solid support. When composed, the plurality of sample polypeptides may be coupled to the solid support with an overall sample preparation efficiency that may be calculated based upon a separation efficiency and a coupling efficiency, where the separation efficiency may be calculated as a function of the plurality of separation standard polypeptides, and wherein the coupling efficiency is calculated as a function of the plurality of coupling standard polypeptides. In some configurations, the overall sample preparation efficiency is linearly proportional to the separation efficiency, the coupling efficiency, or a combination of both (e.g., α is 1 in Equation 1). In some configurations, the overall sample preparation efficiency is non-linearly proportional to the separation efficiency, the coupling efficiency, or a combination of both (e.g., α is greater or less than 1 in Equation 1).

[0124] A polypeptide array may comprise an assay internal standard, in which the assay intenal standard is configured to provide information on the outcome of the assay or a step thereof. For example, a polypeptide assay utilizing a denaturation step may include a denaturation internal standard comprising a ladder of polypeptides containing a same epitope with differing degrees of epitope accessibility (i.e., ranging from fully buried within a tertiary structure to exposed at the surface), in which success of the denaturation step is determined by relatively uniform detection of the same epitope for each polypeptide of the ladder of polypeptides. In another example, an internal standard may comprise a protein containing one or more disulfide bridges, in which presence or absence of a disulfide bridge (e.g., due to an oxidation or reduction step of a polypeptide assay) is determined by detection or lack thereof of a known epitope buried or exposed by the presence or absence of the disulfide bridge. In another example, a fluorosequencing assay may comprise an internal standard peptide for each sequenced amino acid that is configured to provide information on the rate of false positive or false negative detections of the sequenced amino acid.

[0125] An internal standard may comprise a plurality of polypeptides, in which the plurality of polypeptides is characterized by a range of chemical, physical, and / or biological diversity, such as epitope diversity (e.g., all or a subset of all 8000 possible trimer epitopes), net electrical charge diversity (e.g., ranging from positive to negative net charge), mass diversity (e.g., ranging from peptide to ultra-high molecular weight), hydrodynamic radius diversity, species diversity (e.g., different variants of a same polypeptide from different host organisms), proteoform diversity (e.g., all known proteoforms of a polypeptide), genetic diversity (e.g., all or a subset of all polypeptides derived from all single-nucleotide polymorphisms of a gene), or any other conceivable measure of chemical, physical, or biological diversity.

[0126] An internal standard may comprise a plurality of polypeptides, in which the plurality of polypeptides comprises an affinity agent characterization standard. An affinity agent characterization standard may comprise a plurality of polypeptides that are utilized to measure and / or characterize a binding property of an affinity agent, such as a binding specificity, binding affinity, and / or binding promiscuity. An affinity agent characterization standard may comprise a plurality of polypeptides that is utilized across a plurality of assays to provide increased characterization of a known affinity agent or initial characterization of a new or unknown affinity agent.

[0127] An internal standard may comprise a quantitation standard. A quantitation standard may be configured to provide a measure of dynamic range for a polypeptide assay. For example, a quantitation standard may be used to determine a dynamic range of a single-molecule polypeptide assay (e.g., polypeptide identification, polypeptide sequencing, etc.). A quantitation standard may comprise a standard polypeptide with a known concentration, in which the standard polypeptide has an expected quantity on a polypeptide array when deposited. For example, a quantitation may comprise a standard polypeptide whose expected abundance on a polypeptide array is expected to match a particular dynamic range of polypeptides from a sample (e.g., single molecule, tens of molecules, hundreds of molecules, etc.). In some cases, a quantitation standard may comprise a concentration ladder of polypeptides, in which each type of polypeptides in the ladder of polypeptides is used to confirm the detection sensitivity of a particular portion of a dynamic range for detection. In some cases, a quantitation standard may comprise a high-concentration polypeptide that provides an alternative form of quantitation. For example, a polypeptide array may comprise a high-concentration quantitation standard that can be quantitated by an alternative assay such as a Western Blot. In some cases, a quantitation standard may comprise a fluorescent protein (e.g.,, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.). A fluorescent protein may be conjugated or coupled to a quantitation standard polypeptide to provide a detectable label that makes the quantitation standard polypeptide readily identifiable on a polypeptide array.

[0128] A polypeptide array may comprise a fraction of internal standard moieties, as measured by a fraction of array addresses containing an internal standard moiety, or as measured by a fraction of internal standard moieties relative to all moieties coupled to a polypeptide array. A polypeptide array may have an internal standard fraction of at least about 0.000000001, 0.000000005, 0.00000001, 0.00000005, 0.0000001, 0.0000005, 0.000001, 0.000005, 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 0.9, or greater than 0.9. Alternatively or additionally, a polypeptide array may have an internal standard fraction of no more than about 0.9, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, 0.00001, 0.000005, 0.000001, 0.0000005, 0.0000001, 0.00000005, 0.00000001, 0.000000005, 0.000000001, or less than 0.000000001.Polypeptide Isolation

[0129] A sample may be processed or prepared to isolate a polypeptide fraction from a sample. Methods and compositions may be exemplified below with reference to proteins; however, it will be understood that the examples can be extended to peptides or other polypeptides. A sample may be processed to isolate some or all available polypeptides within a sample. A sample may be processed to isolate a subset of all available polypeptides within a sample. A sample with unknown characteristics may be processed or prepared according to a polypeptide isolation method even if no polypeptides are found to exist in the sample after characterization. For example, a sterilized instrument may be swabbed for residual proteins, with the swabbings to be prepared and analyzed for possible protein contamination.

[0130] A sample preparation process may isolate or capture polypeptides from a sample with a known efficiency. A sample preparation process may isolate about 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or more than 99.999% of the available polypeptides within a sample on a molar or mass basis. A sample preparation process may isolate at least about 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or more than about 99.999% of the available polypeptides within a sample on a molar or mass basis. Alternatively or additionally, a sample preparation process may isolate no more than about 99.999%, 99.99%, 99.9%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001% or less than about 0.000001% of the available polypeptides within a sample on a molar or mass basis.

[0131] A sample preparation process may isolate or capture a subset of polypeptides from a sample with a known efficiency. A subset of polypeptides may comprise a specific or targeted group of polypeptides, e.g., membrane proteins, soluble proteins, nuclear proteins, cytoplasmic proteins, extracellular proteins, or blood plasma proteins. A sample preparation process may isolate about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or more than 99.999% of a subset of available polypeptides within a sample on a sequence composition, molar or mass basis. A sample preparation process may isolate at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or more than about 99.999% of a subset of available polypeptides within a sample on a sequence composition, molar or mass basis. Alternatively or additionally, a sample preparation process may isolate no more than about 99.999%, 99.99%, 99.9%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than about 5% of a subset of available polypeptides within a sample on a sequence composition, molar or mass basis.

[0132] A sample preparation process may isolate or capture a sufficient quantity of polypeptides for a polypeptide assay. An isolated polypeptide fraction may consist of at least about 0.1 picograms (pg), 0.5 pg, 1 pg, 5 pg, 10 pg, 50 pg, 100 pg, 500 pg, 1 nanogram (ng), 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 40 ng, 50 ng, 75 ng, 100 ng, 200 ng, 250 ng, 500 ng, 750 ng, 1 microgram (µg), 5 µg, 10 µg, 20 µg, 50 µg, 100 µg, or more than about 100 µg. Alternatively or additionally, an isolated polypeptide fraction may consist of no more than about 100 µg, 50 µg, 20 µg, 10 µg, 5 µg, 1 µg, 750 ng, 500 ng, 250 ng, 200 ng, 100 ng, 75 ng, 50 ng, 40 ng, 30 ng, 20 ng, 15 ng, 10 ng, 5 ng, 1 ng, 500 pg, 100 pg, 50 pg, 10 pg, 5 pg, 1 pg, 0.5 pg, 0.1 pg, or less than 0.1 pg.

[0133] An isolated polypeptide fraction may have a characterizable polypeptide diversity. The polypeptide diversity of a polypeptide fraction may be characterized as being homogeneous or heterogeneous. A polypeptide fraction with homogeneous polypeptide diversity may be comprised almost exclusively of a single protein species. A polypeptide fraction may be considered to have heterogeneous polypeptide diversity if it consists of more than one polypeptide species. A polypeptide fraction may be considered to have homogeneous polypeptide diversity if it contains a single polypeptide species above a threshold value on a mass or molar basis. A polypeptide fraction may be considered to have heterogeneous polypeptide diversity if it contains no single polypeptide species above a threshold value on a mass or molar basis. A polypeptide fraction with homogeneous polypeptide diversity may contain a single polypeptide species above a threshold value of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 99.999%, 99.9999%, or about 99.99999% on a mass or molar basis. A polypeptide fraction with homogeneous polypeptide diversity may contain a single polypeptide species above a threshold value of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than about 99.99999% on a mass or molar basis. Alternatively or additionally, a polypeptide fraction with homogeneous polypeptide diversity may contain a single polypeptide species above a threshold value of no more than about 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, or less than 90% on a mass or molar basis.

[0134] A sample may undergo one or more purification processes to remove one or more waste fractions comprising impurities (polypeptide or non-polypeptide) or sample fractions that are not of interest, thereby yielding an isolated or purified polypeptide fraction. An isolated polypeptide fraction may be substantially free of impurities. For example, an isolated polypeptide fraction may be substantially free of one, some or all species of impurities set forth herein or known in the art. In some cases an isolated polypeptide fraction may have a substantially reduced amount of one, some or all species of impurities set forth herein or known in the art. The scope of what constitutes an impurity may depend upon the nature of the polypeptide assay to be performed. Impurities may include small molecule components (e.g., amino acids, nucleotides, vitamins, coenzymes, glucose, etc.), other non-polypeptide biomolecules (e.g., lipids, nucleic acids, polysaccharides, etc.), salts, heavy metals, solvents, and non-biological chemicals (e.g., an inorganic impurity, an organic impurity, an acid, a base, a mineral, a polymer, a metal, a semiconductor, a ceramic, etc.). In some cases, an isolated polypeptide fraction may be free of a subset of polypeptides that are not relevant to the characterization to be performed. An isolated polypeptide fraction may be purified to remove unwanted polypeptides, such as degraded proteins, agglomerated proteins, damaged proteins, truncated proteins, low-molecular weight proteins or peptides, high-molecular weight proteins, organelle-specific proteins (e.g., membrane proteins, nuclear proteins, etc.), or specific species of polypeptides that are to be excluded from an analysis. Certain contaminants may be removed by an enzymatic, chemical or physiological method. In some cases, a sample may be prepared for a polypeptide assay with one or more impurities remaining. For example, in certain detection assays, the relative abundance of impurities may be unimportant provided a target polypeptide is still detectable within an impure polypeptide fraction.

[0135] An isolated polypeptide fraction may be analyzed for relative or absolute purity prior to or during a polypeptide assay set forth herein. Relative purity may be considered as the amount of one or more impurities within an isolated polypeptide fraction (e.g., nucleic acid content, metal content). Relative purity may be presented as an absolute amount (e.g., 10 parts per billion (ppb) iron) or an amount relative to a pre-purification level (e.g., iron at 0.001% of pre-purification amount). The pre-purification level can be determined as the amount of the one or more impurities in the original source for the isolated polypeptide fraction (e.g. biological fluid, cell, tissue etc.) or in an initial sample (e.g. blood sample) or extract (e.g. crude cell lysate) of the original source. The pre-purification level can be determined empirically or based on a known or suspected level. A polypeptide fraction may be considered free of an impurity if the impurity is below a detectable threshold for a standard method of measurement. An absolute impurity may be considered as the total amount of impurity in a polypeptide fraction on a dry mass basis (e.g., 1000 parts per million (ppm) or 0.1 wt % impurity) or on a molar basis. Purity of one or more polypeptides in an isolated polypeptide fraction can be determined relative to the amount of one or more standard polypeptide set forth herein.

[0136] An isolated polypeptide fraction may be purified until the abundances of one or more impurities falls below a threshold level. The threshold level may be an impurity level below which the impurity will not substantially interfere with the results of a polypeptide assay. The threshold level of an impurity may be about 0.1 ppb, 1 ppb, 10 ppb, 50 ppb, 100 ppb, 200 ppb, 500 ppb, 1 ppm, 5 ppm, 10 ppm, 50 ppm, 100 ppm, 500 ppm, 1000 ppm, or more than about 1000 ppm. The threshold level of an impurity may be at least about 0.1 ppb, 1 ppb, 10 ppb, 50 ppb, 100 ppb, 200 ppb, 500 ppb, 1 ppm, 5 ppm, 10 ppm, 50 ppm, 100 ppm, 500 ppm, 1000 ppm, or more than about 1000 ppm. Alternatively or additionally, the threshold level of a component may be about 1000 ppm, 500 ppm, 100 ppm, 50 ppm, 10 ppm, 5 ppm, 1 ppm, 500 ppb, 100 ppb, 50 ppb, 10 ppb, 5 ppb, 1 ppb, 0.5 ppb, 0.1 ppb or less than about 0.1 ppb.

[0137] An isolated polypeptide fraction may be purified until the abundance of one, some or all impurities falls below a threshold level. The threshold level may be an impurity level (e.g. a total impurity level) below which the impurities will not substantially interfere with the results of a polypeptide assay. The threshold level of one, some or all impurities may be about 10 weight percent (wt %), 9 wt%, 8 wt%, 7 wt%, 6 wt%, 5 wt%, 4 wt%, 3 wt%, 2 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt%, 0.001 wt%, 0.0005 wt%, 0.0001 wt%, 0.00005 wt%, 0.00001 wt%, or less. Alternatively or additionally, the threshold level of all impurities may be no more than about 10 weight percent (wt %), 9 wt%, 8 wt%, 7 wt%, 6 wt%, 5 wt%, 4 wt%, 3 wt%, 2 wt%, 1 wt%, 0.5 wt%, 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt%, 0.001 wt%, 0.0005 wt%, 0.0001 wt%, 0.00005 wt%, 0.00001 wt%, or less than 0.00001 wt%.

[0138] A polypeptide fraction produced by a sample preparation process may comprise polypeptides of natural or synthetic origin. Polypeptides may contain post-translational modifications or post-synthesis modifications. In some cases, polypeptides may be treated to add or remove post-translational modifications or post-synthesis modifications. Such modifications can be made as part of a sample pretreatment method, such as those set forth elsewhere herein. Post-translational or post-synthesis modifications may include myristoylation, palmitoylation, isoprenylation, prenylation, farnesylation, geranylgeranylation, lipoylation, flavin moiety attachment, Heme C attachment, phosphopantetheinylation, retinylidene Schiff base formation, dipthamide formation, ethanolamine phosphoglycerol attachment, hypusine, beta-Lysine addition, acylation, acetylation, deacetylation, formylation, alkylation, methylation, C-terminus amidation, arginylation, polyglutamylation, polyglyclyation, butyrylation, gamma-carboxylation, glycosylation, glycation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphoate ester formation, phosphoramidate formation, phosphorylation, adenylylation, uridylylation, propionylation, pyrolglutamate formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, S-sulfinylation, S-sulfonylation, succinylation, sulfation, glycation, carbamylation, carbonylation, isopeptide bond formation, biotinylation, carbamylation, oxidation, reduction, pegylation, ISGylation, SUMOylation, ubiquitination, neddylation, pupylation, citrullination, deamidation, elminylation, disulfide bridge formation, proteolytic cleavage, isoaspartate formation, racemization, and protein splicing.

[0139] A sample preparation process that is used to isolate a polypeptide fraction from a sample may vary depending upon the type of sample and the specific assay to be performed. In some cases, a sample may be prepared for a proteomic-scale assay, thus employing high-efficiency capture of the full proteome, or a substantial portion thereof, within the sample. In other cases, a sample may be prepared for a specific fraction of polypeptides, thus utilizing high-efficiency capture of that fraction of the polypeptides. Likewise, in some cases, a sample may be prepared for single-molecule quantitation, benefitting from high-purity polypeptide fractions. In other cases, a sample may be prepared to only determine the presence of one or more biomarkers, where the presence of impurities is of lesser concern. As such, sample preparation procedures may be varied with regard to the method of isolating polypeptides and the method of removing impurities.

[0140] Sample preparation to isolate or separate protein fractions may involve several steps such as those set forth herein. Certain steps that are set forth herein may be omitted, varied or reordered depending upon the nature of the sample to be prepared. For example, steps used to obtain a protein sample from a fabric for forensic purposes may be different than the steps used to obtain a full proteomic protein sample from a pellet of bacteria. There may be different methods of protein isolation or separation used for biological samples (e.g., cells, blood) as opposed to curated samples (e.g., textiles, rocks). There may also be different methods of protein isolation or separation for protein fractions derived from solid media (e.g., tissue, single cells, fabrics, filters) as opposed to liquid media (bodily fluids, water samples). A standard polypeptide can be added prior to or during any of the steps set forth herein or known in the art. The added standard polypeptide can be used to evaluate a characteristic or quantity of one or more polypeptides obtained using the step.

[0141] A procedure for protein isolation or separation from a biological sample may be configured to suit a particular sample, for example, based on characteristics such as whether polypeptides are possibly localized on or within a biological compartment (e.g., an organism, tissue, cell or organelle) or are free in solution (e.g., proteins in blood plasma or other biological fluid). For example, a blood sample may be prepared differently if all polypeptides (including those in red or white blood cells) are to be captured rather than only cell free polypeptides from blood plasma. FIG. 2 depicts a flowchart for a general method of isolating polypeptide fractions from biological samples. The starting point in the process may depend upon whether polypeptides are to be derived from a sample containing polypeptides within a solid medium 200 or a sample in which free-solution polypeptides are to be retained 201. For a sample in which polypeptides are to be derived from within a solid medium 200, the sample 200 may be treated to break up or disrupt a cell wall if one is present. A cell wall may be disrupted 210 mechanically, enzymatically, or chemically. A cell wall may be disrupted 210 in the presence of a detergent. A cell wall may be disrupted 210 in the presence of a standard polypeptide, such as a cell wall disruption standard polypeptide. Also, for a sample in which polypeptides are to be derived from within a solid medium 200, the sample 200 may be treated to lyse or disrupt a cell membrane if one is present. A cell membrane may be disrupted 220 mechanically (e.g., shearing, french press or sonication) or chemically (e.g., exposure of the cell to hypotonic or hyperionic solution, pH extremes, lytic enzymes or the like). A cell membrane may be disrupted 220 in the presence of a detergent. A cell membrane may be disrupted 220 in the presence of a standard polypeptide, such as a membrane disruption standard polypeptide. A cell wall or cell membrane can be disrupted using methods known in the art such as those set forth in Scopes, Protein Purification: Principles and Practice, 3rd Ed., Springer Advanced Texts in Chemistry (1993); Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1998).

[0142] After a sample 200 has been sufficiently disrupted, the resulting material may include fragments of cell walls, cell membranes, nuclear membranes, organelles, capsids, nucleic acids, and other biological materials, as well as polypeptides that are suspended in solution. It may be necessary to extract additional polypeptides 230 from fragments of the disrupted sample 200 (e.g., membrane proteins, organelle proteins). The additional polypeptides may be extracted 230 in the presence of a detergent. The additional polypeptides may be extracted 230 in the presence of a standard polypeptide, such as an extraction standard polypeptide. After the extraction of additional polypeptides 230, a polypeptide fraction may be separated, isolated, or precipitated 240 from the non-polypeptide fragments of the sample 230. In some cases, the non-polypeptide fragments may be retained for one or more additional rounds of polypeptide separation and extraction. Any suitable method may be utilized for polypeptide separation, isolation or precipitation, including, for example, precipitation such as high salt or organic solvent based separations; centrifugation to separate membranes and insoluble matter from soluble matter; filtration to separate matter in solution from insoluble matter; liquid-phase extraction to separate matter based on solvation properties; solid-phase extraction to separate matter based on affinity for a solid support having predefined chemical properties; chromatography, such as affinity chromatography to separate matter based on affinity for a particular analyte, ionic exchange chromatography to separate matter based on ionic charge, size exclusion chromatography to separate matter based on size; hydrophobic exchange chromatography; reverse phase chromatography and other methods known in the art such as those set forth in Scopes Protein Purification: Principles and Practice, 3rd Ed., Springer Advanced Texts in Chemistry (1993). Separation, isolation, or precipitation 240 of a polypeptide fraction may occur in the presence of a detergent. Separation, isolation, or precipitation 240 of a polypeptide fraction may occur in the presence of a standard polypeptide, such as a separation standard polypeptide, isolation standard polypeptide, or precipitation standard polypeptide.

[0143] A particularly useful method for separating polypeptides of interest from other sample components is selective adsorption on insoluble supports such as resins, beads, particles or the like. For example, particles can be used to adsorb polypeptides based on attraction between the polypeptides and the surface of the nanoparticles. Polypeptides in a biological fluid, such as plasma, can form a polypeptide layer that coats the particles on the particle surface, forming a protein corona. The composition and quantity of corona polypeptides is a function of the physicochemical properties of the particles. Particles can be sleeted or engineered with surface properties that reproducibly produces variation in the corona in terms of identity and / or quantity of polypeptides. Particles can be contacted with a sample such that polypeptide coronas form on the particles. The polypeptide coated particles can be separated from the sample (e.g. a magnet can be used to attract paramagnetic or magnetic particles, gravity can be used to attract dense particles etc.) and then the polypeptides can be released for further processing. An exemplary super- paramagnetic iron oxide nanoparticle (SPION) can be coated with a thin layer of silica by a modified Stöber process using tetraethyl orthosilicate (TEOS). Other exemplary SPIONs can be coated with poly(dimethylaminopropyl methacrylamide) or poly(ethylene glycol). These and other particles, methods for making the particles and methods for their use are set forth in Blume et al. Nat. Commun. 11, 3662 (2020); US Pat. Nos. 10,866,242, 10,272,050, or 10,022,334; or US Pat. App. Pub. Nos. 2019 / 0350870 A1, 2020 / 0138728 A1 or 2020 / 0206145 . Another useful capture substrate is pyridinyl carboxaldehyde derivative attached to a resin or other particle. See, for example, Howard et al. ACS Chem. Biol. 15, 1401-1407 (2020).

[0144] The particles can be attached to a standard polypeptide and the standard polypeptide can be used to evaluate a characteristic of polypeptides that are known or expected to form a corona and / or the standard polypeptide can be used to evaluate a characteristic of polypeptides that are known or expected to be released from the corona. In some configurations, particles having different surfaces can be attached to unique standard polypeptides that indicate the type of surface the particle has, the type of polypeptide that is expected to form a corona on the particle, or the type of polypeptide that is expected to be released from the corona. The particles can be attached to standard polypeptides using any of a variety of attachment mechanisms known in the art or set forth herein, including for example, covalent bonding, non-covalent bonding, adsorption, or the like. A standard polypeptide can be attached to a particle before or after the particle being contacted with a sample. In some cases, a standard polypeptide can be present in a sample that is contacted with the particle, such that the standard polypeptide attaches to the particle, for example by adsorbing with sample polypeptides to form a polypeptide corona.

[0145] Following separation, isolation, or precipitation 240 of a polypeptide fraction, the polypeptide fraction may be further purified 250 to remove residual impurities, yielding a final purified polypeptide fraction 260. Impurities removed 250 may include metals, salts, or remaining non-polypeptide biological fragments. Exemplary purification techniques may include, for example, those set forth above or in Scopes Protein Purification: Principles and Practice, 3rd Ed., Springer Advanced Texts in Chemistry (1993).

[0146] A purified polypeptide fraction 260 may be subjected to one or more additional rounds of purification to arrive at a substantially purified polypeptide fraction 260. The skilled person will recognize that certain steps of the process shown in FIG. 2 can be rearranged or run in parallel. For example, free-solution polypeptides may be separated 240 from non-polypeptide biological materials before polypeptides have been extracted from the non-polypeptide biological materials. Separation of polypeptides from the non-polypeptide biological materials may occur in parallel after separation 240, with extracted polypeptides combined with free-solution polypeptides before or after final purification 250.

[0147] FIG. 2 also depicts an exemplary separation process for samples in which free-solution polypeptides are to be selectively retained 201. The sample 201 may be processed 235 to separate unwanted components from the free-solution polypeptides. Methods such as centrifugation, size-exclusion chromatography, liquid chromatography, other techniques set forth herein, or a combination thereof, may be utilized to separate unwanted components (e.g., cells) from the free-solution polypeptides. This separation may be utilized to remove components that greatly differ in size from the free-solution polypeptides. The processing 235 may occur in the presence of a detergent. The processing 235 may occur in the presence of a standard polypeptide, such as a separation standard polypeptide. After processing 235 the sample 201, the purified fraction may be subjected to separation, isolation, or precipitation 240 of a polypeptide fraction. Any suitable method may be utilized for polypeptide separation, isolation or precipitation, including methods set forth above or elsewhere herein. Separation, isolation, or precipitation 240 of a polypeptide fraction may occur in the presence of a detergent. Separation, isolation, or precipitation 240 of a polypeptide fraction may occur in the presence of a standard polypeptide, such as a separation standard polypeptide, isolation standard polypeptide, or precipitation standard polypeptide. Following separation, isolation, or precipitation 240 of a polypeptide fraction, the polypeptide fraction may be further purified 250 to remove residual impurities, yielding a final purified polypeptide fraction 260. Impurities removed 250 may include metals, salts, or remaining non-polypeptide biological fragments. Exemplary purification techniques may include methods set forth above or elsewhere herein. A purified polypeptide fraction 260 may be subjected to one or more additional rounds of purification to arrive at a substantially purified polypeptide fraction 260.

[0148] FIG. 3 depicts a process for isolating or separating a polypeptide fraction from a curated sample. The procedure for polypeptide isolation or separation from a curated or environmental sample may be configured based upon whether the sample 300 is in a solid or liquid medium. If the sample 300 comprises a liquid medium, the sample may be moved directly to a polypeptide extraction process 320. If the sample 300 comprises a solid medium, the processing of the sample may be configured based upon whether the sample 300 may be altered. For example, a sample may be an artifact, forensic specimen or paleontological specimen. In another example, a polypeptide sample may be isolated from a solid surface such as an air filter or liquid filter without damaging the filter. If the sample can be altered, it may undergo a preliminary processing 305 that may improve the extraction of polypeptide from the solid sample. The preliminary processing 305 may include crushing, grinding, pulverization, heating, cooling, steam treatment, or chemical dissolution. After an optional preliminary processing step 305, the sample 300 may be transferred into a solvent extraction process 310. The solvent extraction process 310 may involve the use of one or more solvents to loosen or solvate polypeptides bound to the solid medium. The solvent extraction process 310 may involve additional extraction enhancers such as heat, denaturants, or mixing. A solvent mixture for a solvent extraction process 310 may include a detergent. A solvent mixture for a solvent extraction process 310 may include a standard polypeptide, such as an extraction standard polypeptide. The solvent extraction process 310 may result in a liquid medium containing free-solution polypeptides. A liquid medium containing free-solution polypeptides may be subjected to a polypeptide precipitation process 320. The free-solution polypeptides in the liquid medium may be separated, isolated, or precipitated 320 from the liquid medium of the sample. In some cases, the liquid medium may be retained for one or more additional rounds of polypeptide separation and extraction. Any suitable method may be utilized for polypeptide separation, isolation or precipitation, including the methods listed above. Separation, isolation, or precipitation 320 of a polypeptide fraction may occur in the presence of a detergent. Separation, isolation, or precipitation 320 of a polypeptide fraction may occur in the presence of a standard polypeptide, such as a separation standard polypeptide, isolation standard polypeptide, or precipitation standard polypeptide. Following separation, isolation, or precipitation 320 of a polypeptide fraction, the polypeptide fraction may be further purified 330 to remove residual impurities, yielding a final purified polypeptide fraction 340. Impurities removed 330 may include metals, salts, or remaining non-polypeptide biological fragments. Exemplary purification techniques may include the methods listed above. A purified polypeptide fraction 340 may be subjected to one or more additional rounds of purification to arrive at a final purified polypeptide fraction 340.

[0149] Polypeptide fractions may be prepared to remove certain polypeptides. Polypeptides may be separated or excluded from a polypeptide fraction if they are larger than a particular size. Polypeptides may be separated or excluded from a polypeptide fraction if they are smaller than a particular size. A polypeptide fraction may be treated to reduce the size of large polypeptides, for example by protease treatment or hydrolysis. Polypeptides may be separated or excluded from a polypeptide fraction based on size by methods such as density-gradient centrifugation, size-exclusion chromatography, filtration, dialysis, gel electrophoresis or other methods set forth herein for polypeptide separation. A polypeptide fraction may be treated to generate a peptide fraction, wherein the average length of the peptides in the peptide fraction is substantially smaller than the average length of the polypeptides in the polypeptide fraction. Peptides generated from a polypeptide fraction may have an average size that is at least 50%, 25%, 10%, 5%, or less of the average size of the polypeptides in the polypeptide fraction. A polypeptide fraction may undergo one or more processes to alter the size distribution of a polypeptide fraction, for example protease treatment followed by a separation process such as size exclusion chromatography.

[0150] A sample comprising a plurality of polypeptides may be processed or prepared to produce one or more polypeptide fractions. A sample comprising a plurality of polypeptides may be processed or prepared to produce a plurality of polypeptide fractions. A plurality of polypeptide fractions may be produced such that each polypeptide fraction has a different physical attribute. For example, polypeptide fractions may be distinguished by comprising polypeptides of differing types (e.g., extracellular, membrane, cytoplasmic, etc.), differing size ranges, differing chemical properties (e.g., hydrophobicity), differing isoelectric points, polarity, pKa etc. A plurality of polypeptide fractions may be produced by separating fractions on the basis of a polypeptide property, such as polypeptide size, polypeptide isoelectric point, polarity, pKa, polypeptide hydrophobicity, polypeptide hydrodynamic radius, polypeptide state, or the presence or absence of post-translational modifications. A sample may be separated into about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more than 100 polypeptide fractions. A sample may be separated into at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more than 100 polypeptide fractions. Alternatively or additionally, a sample may be separated into no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less than 2 polypeptide fractions.

[0151] The preparation of polypeptide fractions from a sample may involve one or more separation processes that isolate or purify polypeptides from non-polypeptide sample components. The preparation of polypeptide fractions from a sample may involve one or more separation processes that separate a first plurality of polypeptides from a second plurality of polypeptides. Polypeptide fractions may be produced by any suitable method. A polypeptide fraction separation process may comprise a fractionation method, a targeting method, or a combination thereof. Fractionation methods may include bulk methods that separate polypeptides based upon polypeptide properties. Exemplary fractionation methods may include chromatographic methods (e.g., size exclusion chromatography, reverse-phase liquid chromatography, ion exchange chromatography, hydrophobic exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, hydrophilic interaction liquid chromatography, mixed-mode chromatography, 2-dimensional liquid chromatography, etc.), centrifugation methods (e.g., ultracentrifugation, density gradient centrifugation, etc.), electrophoretic methods (e.g., 1-D or 2-D SDS-page gel electrophoresis, isoelectric focusing, free-flow electrophoresis, pulsed-field electrophoresis, field flow fractionation, etc.), solid phase adsorption, solid phase absorption, and precipitation methods (e.g., acetone precipitation, ethanol precipitation, methanol precipitation, salting-in, salting-out, hydrophilic polymer precipitation, polyelectrolyte flocculation, polyvalent cation precipitation, etc.). Targeting methods may include any separation method that separates a polypeptide species or group of polypeptide species from a plurality of polypeptide species with a desired specificity. Targeting methods may include the use of capture reagents or pull-down reagents to specifically target and isolate target polypeptides within a plurality of polypeptides. In some cases, a targeting method may utilize a capture reagent or a pull-down reagent that specifically couples with or binds to a subset of polypeptides or polypeptide species within a plurality of polypeptides or polypeptide species (e.g., antibodies, aptamers, etc.). In some cases, a targeting method may utilize a capture reagent or a pull-down reagent that is specifically coupled with or bound to a subset of polypeptides or polypeptide species within a plurality of polypeptides or polypeptide species (e.g., binding ligands, binding substrates).

[0152] In certain polypeptide assays, a polypeptide fraction may be displayed as an array of polypeptides on a solid support. A separation method for producing polypeptides as an array on a solid support may be configured to occur in the presence of the solid support. In some configurations, a solid support may be contained within a fluidic device such as a flow cell, microfluidic device, cartridge, or chip that is configured to facilitate a separation process for polypeptide sample preparation, such as a polypeptide fractionation method or a polypeptide targeting method. In some configurations, a solid support may be provided with a plurality of capture agents that are coupled to a surface of the solid support and are configured to couple to or bind with a polypeptide or polypeptide species. In other configurations, a plurality of polypeptides may be contacted with a pull-down reagent that is configured to couple to a solid support and is further configured to couple to or bind with a polypeptide or polypeptide species.

[0153] A polypeptide fraction may be produced by a targeting separation method. A targeting separation method may comprise the steps of: 1) contacting or combining a sample with a targeting agent that is configured to bind a sample polypeptide of a plurality of sample polypeptides; 2) coupling a sample polypeptide from the plurality of sample polypeptides to the targeting agent; 3) separating a coupled sample polypeptide and targeting agent from the separation mixture; and optionally 4) binding the sample polypeptide to a solid support. The targeting agent may comprise a capture reagent or a pull down reagent. In some configurations, the separation mixture may comprise a plurality of sample polypeptides and a plurality of separation standard polypeptides. In some configurations, the separation mixture may comprise a plurality of sample polypeptides, where the plurality of sample polypeptides comprises target sample polypeptides and non-target sample polypeptides. The targeting agent may be configured to couple to or bind with target sample polypeptides of the plurality of sample polypeptides. In some configurations, the targeting agent may comprise an anchoring group that is configured to couple with a solid support, or may be configured to couple with an anchoring group that is configured to couple with a solid support.

[0154] A targeting agent may be directly contacted with a sample to couple a polypeptide from the sample. For example, a targeting agent may be contacted with a non-disrupted cell sample or a non-disrupted tissue sample (i.e., not homogenized or lysed) to directly remove a polypeptide from the cell sample or the tissue sample without further disruption of the cell or tissue. In some cases, a targeting separation method may comprise the steps of: 1) contacting or combining a sample with a targeting agent that is configured to bind a polypeptide; 2) coupling a polypeptide from the sample to the targeting agent; 3) separating a coupled polypeptide and targeting agent from the sample; and optionally 4) binding the polypeptide to a solid support. The targeting agent may comprise a capture reagent or a pull down reagent. In some configurations, the sample may comprise a plurality of sample polypeptides and a plurality of separation standard polypeptides. In some configurations, the sample may comprise a plurality of sample polypeptides, where the plurality of sample polypeptides comprises target sample polypeptides and non-target sample polypeptides. The targeting agent may be configured to couple to or bind with target sample polypeptides of the plurality of sample polypeptides. In some configurations, the targeting agent may be configured to couple with a solid support, or may be configured to couple with an anchoring group that is configured to couple with a solid support.

[0155] FIGs. 4A - 4F depict various methods for utilizing targeting to isolate a polypeptide fraction from a sample. FIGs. 4A - 4B depict a pull down method for targeting polypeptides in a plurality of polypeptides derived from a sample. The plurality of polypeptides may comprise target polypeptides 420 that contain a target moiety 425 (e.g., an epitope, an amino acid sidechain, a functional group, reactive handle, oligonucleotide, etc.) that is configured to couple to a capture reagent 440 comprising a coupling group 445 (e.g., affinity reagent, complementary functional group, complementary reactive handle, complementary oligonucleotide, etc.) that is configured to couple to the target moiety 425. The plurality of polypeptides may also comprise non-target polypeptides 430. The capture reagent 440 may be configured to couple to a solid support 410. The plurality of polypeptides may be contacted with a plurality of capture reagents 440, thereby allowing the coupling groups 445 of the capture reagents 440 to couple the target moiety 425 of the target polypeptides 420. The capture reagents 440 that are coupled to the target polypeptides 420 may be coupled to the solid support 410, thereby separating the target polypeptides 420 from the non-target polypeptides 430. The non-target polypeptides 430 may be rinsed or washed from the solid support 410, leaving behind a plurality of target polypeptides 420 coupled to the solid support 410.

[0156] FIGs. 4C - 4D depict a pull down method for targeting polypeptides in a plurality of polypeptides derived from a sample. The plurality of polypeptides may comprise target polypeptides 420 that contain a target moiety 425 (e.g., an epitope, an amino acid sidechain, a functional group, reactive handle, oligonucleotide, etc.) that is configured to couple to a capture reagent 440 comprising a coupling group 445 (e.g., affinity reagent, complementary functional group, complementary reactive handle, complementary oligonucleotide, etc.) that is configured to couple to the target moiety 425. The plurality of polypeptides may also comprise non-target polypeptides 430. The capture reagent 440 may be coupled to a solid support 410. The plurality of polypeptides may be contacted with a plurality of capture reagents 440 coupled to the solid support, thereby allowing the coupling groups 445 of the capture reagents 440 to couple the target moiety 425 of the target polypeptides 420, thereby separating the target polypeptides 420 from the non-target polypeptides 430. The non-target polypeptides 430 may be rinsed or washed from the solid support 410, leaving behind a plurality of target polypeptides 420 coupled to the solid support 410.

[0157] FIGs. 4E - 4G depict an indirect pull down method for targeting polypeptides in a plurality of peptides derived from a sample. The plurality of polypeptides may comprise target polypeptides 420 that contain a target moiety 425 (e.g., an epitope, an amino acid sidechain, a functional group, reactive handle, oligonucleotide, etc.) that is configured to couple to a capture reagent 441 comprising a coupling group 445 (e.g., affinity reagent, complementary functional group, complementary reactive handle, complementary oligonucleotide, etc.) that is configured to couple to the target moiety 425. The plurality of polypeptides may also comprise non-target polypeptides 430. The capture reagent 441 may be coupled to a solid support 410. The plurality of polypeptides may be contacted with a plurality of transfer pull down reagents 440. In some configurations, the transfer pull down reagents 440 may comprise a binding target 448 (e.g., a small molecule substrate, a nucleic acid, a polypeptide, etc.) that binds the target polypeptide 420. The binding target 448 may bind a target polypeptide 420 then be transported to a surface of the solid support 410 where the target polypeptides may contact the capture reagents 441. The coupling group 445 of the capture reagent 440 may couple to the target moiety 425 of the target polypeptide 420, thereby separating the target polypeptide 420 from the non-target polypeptides 430. The non-target polypeptides 430 may be rinsed or washed from the solid support 410, leaving behind a plurality of target polypeptides 420 coupled to the solid support 410.

[0158] Targeting assays that utilize a capture agent or pull down reagent may comprise any suitable components for the selective removal of target polypeptides from a plurality of sample polypeptides. The capture agent or pull down agent may comprise a retaining component and one or more coupling components. The retaining component may provide a structure or scaffold for providing a necessary utility to a capture agent or a pull down reagent. A retaining component may comprise a body that can be free in a liquid medium, such as a particle or nanoparticle. In some cases, a retaining component may comprise a particle such as an organic nanoparticle (e.g., polymers, biopolymers, dendrimers, hydrogels, micelles, liposomes, colloids, carbon nanoparticles, etc.), an inorganic nanoparticle (e.g., SiO 2 nanoparticles, Fe 2 O 3 nanoparticles, quantum dots, etc.), or a biomolecule (e.g., a cellulose nanoparticle, a structured nucleic acid particle, a polypeptide, etc.). In some cases, a polymer retaining component may comprise an anionic polymer or a cationic polymer. In some cases, a biopolymer retaining component may comprise a polysaccharide, a polypeptide (e.g., a native or engineered protein), or an oligonucleotide. In some cases, a retaining component may comprise a structured nucleic acid particle (SNAP), such as a nucleic acid origami or a nucleic acid nanoball. A retaining component may further comprise a layer, shell, or coating that alters the properties of the retaining component. For example, a retaining component particle may comprise a metal, polymer or biopolymer coating, or a surface layer of functional groups that are configured to permit coupling or conjugation of other moieties to the particle surface. A retaining component, such as a polymer or biopolymer, may be structured to have an increased surface electrical charge. For example, a protein may be modified to have a larger surface electrical charge than a native protein. A retaining component may be configured to couple to a solid support by a covalent or non-covalent interaction. In some configurations, a capture reagent or pull down reagent may comprise an anchoring group that is configured to couple a polypeptide to a solid support. A retaining component may comprise a fixed support or phase, such as a resin. A fixed retaining component may be configured as, for example, an affinity chromatography medium.

[0159] A capture agent or pull down reagent may further comprise coupling groups that are configured to couple a polypeptide to the capture agent or pull down reagent. Coupling groups may be configured to form a covalent or non-covalent interaction with a polypeptide. A coupling group may comprise a functional group or reactive handle that is configured to form a covalent bond with a functional group or reactive handle on a polypeptide. A reactive handle on a capture reagent or pull down reagent may comprise a functional group that is configured to undergo a click reaction. A coupling group may include a group that is configured to form a non-covalent interaction with a polypeptide or a group coupled to a polypeptide. Exemplary non-covalent interactions may include nucleic acid oligonucleotide hybridization and receptor-ligand or binder-small molecule pairs, such as streptavidin-biotin, glutathione-glutathione-S-transferase, maltose-maltose binding protein, and chitin-chitin binding protein. In some configuration, a coupling group may comprise a binding ligand that selectively scavenges targeted polypeptides from a sample. For example, polypeptides with specific binding functions, such as G-proteins, transcription factors, promoter polypeptides, repressor polypeptides, and histones, may be targeted by capture reagents or pull down reagents displaying nucleic acid sequences that are specific to the binding polypeptide. A targeting agent, such as a capture reagent or pull down reagent, may be configured to couple to one or more internal standard polypeptides of a plurality of internal standard polypeptides, such as sample collection standard polypeptides, sample handling standard polypeptides, separation standard polypeptides, functionalization standard polypeptides, coupling standard polypeptides, and attachment standard polypeptides.

[0160] A targeting agent, such as a capture reagent or pull down reagent, may be configured to couple with a sample polypeptide by a chemical linkage, such as a chemical bond, a catalyzed bond, or an enzymatically-catalyzed linkage. A capture reagent or pull down reagent may comprise one or more chemically-modifying groups, such as post-translational modification groups (e.g., SUMO, ubiquitin, acetyl, formyl, phosphate, glycans, isoprenoids, etc.) that are coupled or conjugated to the capture reagent or pull down reagent. The capture reagent or pull down reagent comprising the chemically-modifying group may be combined with a sample polypeptide in the presence of an enzyme (e.g., ubiquitin-E3 ligase, acetylase, isoprenoid transferase, etc.) that is configured to catalyze attachment of the sample polypeptide to the chemically-modifying group at any available attachment site. A sample polypeptide that is available for a chemical modification may become linked to the capture reagent or pull down reagent, thereby permitting it to be isolated from a sample.

[0161] A capture reagent or pull down reagent for a targeting separation method may be configured to couple to one or more polypeptides. A capture reagent or pull down reagent may be configured to couple to a plurality of polypeptides. A capture reagent or pull down reagent for a targeting separation method may be configured to couple to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 polypeptides. A capture reagent or pull down reagent for a targeting separation method may be configured to couple to at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 polypeptides. Alternatively or additionally, a capture reagent or pull down reagent for a targeting separation method may be configured to couple to no more than about 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or less than 2 polypeptides.

[0162] In some cases, a capture reagent or a pull down reagent may be configured to act as a transfer agent or intermediary for targeting polypeptides during a separation process. A transfer agent or intermediary may couple to a polypeptide, then transfer the polypeptide to an anchoring group that is coupled to a solid support, or configured to be coupled to a solid support. FIGs. 4E - 4G depict an example of a transfer agent. A transfer agent or intermediary may release a polypeptide to the anchoring group, or may be configured to bind to the anchoring group, thereby coupling the polypeptide to the anchoring group.

[0163] A capture reagent or a pull down reagent utilized for a targeting separation method may degrade during usage. For example, a capture agent utilizing a nucleic acid capture agent may be degraded by the presence of particular enzymes when contacted with some cellular or lysate samples (e.g., restriction enzymes). A capture reagent or a pull down reagent may be designed to degrade during usage. Degradation can optionally be initiated by application of a degrading reagent (e.g. oxidant, reducing agent, enzyme) or stimulus (e.g. light or heat). For example, a capture agent comprising photocleavable linkers may degrade into subcomponents upon application of radiation of a particular wavelength. In another example, a nucleic acid-based capture agent can be degraded by an enzyme such as a nuclease or by a chemical agent such as piperidine. The degradable capture reagent or pull down reagent may be degraded before, during, or after contact with a plurality of polypeptides. The degradable capture reagent or pull down reagent may be degraded before, during, or after contact with a solid support or a plurality of anchoring groups.

[0164] FIGs. 5A - 5H depict various configurations of capture reagents with degradable properties. FIG. 5A depicts a capture reagent comprising a plurality of subunits 540 (e.g., a structured nucleic acid particle, a cluster of linked nanoparticles). Each subunit 540 of the plurality of subunits comprises a coupling group 545 that is configured to couple with a polypeptide and a coupling group 548 that is configured to be coupled with a solid support or an anchoring group. The capture reagent comprising the plurality of subunits 540 is contacted with a plurality of polypeptides 520 comprising coupling groups 525 that are configured to be coupled by the polypeptide coupling groups 545 of the capture reagent. FIG. 5B shows the capture reagent subunits 540 with coupled polypeptides 520 after the capture reagent has undergone a degradation process (e.g., enzymatic degradation, photolytic degradation, chemical degradation, etc.). The polypeptide-coupled subunits 540 may be contacted with a solid support 510 comprising a plurality of anchoring groups 530 with coupling groups 535 that are configured to couple with the coupling group 548 of the capture reagent subunit 540. As shown in FIG. 5C, after contacting, the subunits 540 may be coupled to the anchoring groups 530, thereby coupling the polypeptides 520 to the solid support 510.

[0165] FIGs. 5D - 5F show an example of a capture reagent or pull down reagent for a targeting separation method with a designed degradable structure. As shown in FIG. 5D, the capture reagent comprises a structured nucleic acid particle 550 (e.g., a DNA nanoball) comprising surface-displayed coupling groups 558 that are configured to couple with polypeptides and surface-displayed coupling groups 545 that are configured to be coupled with a solid support or an anchoring group. FIG. 5E shows an exploded view of the particle when the scaffold of the capture reagent or pull down reagent is unfolded. The structured nucleic acid particle 550 comprises a plurality of concatemerized subunits 551, with each subunit 551 comprising a polypeptide coupling group 558 and a coupling group 545 that is configured to be coupled with a solid support or an anchoring group. The concatemerized subunits 551 are linked by a degradation site 552 (e.g., a restriction site, a photocleavable linker, etc.) that is configured to be cleaved under suitable conditions. FIG. 5F depicts a capture reagent 550 contacted with a plurality of polypeptides 520, thereby coupling the polypeptides to the subunits 551. The subunits each comprise a free coupling group 555 that may facilitate attachment of the subunits to a solid support or an anchoring group.

[0166] FIGs. 5G - 5H show an additional example of a capture reagent or pull down reagent for a targeting separation method with a designed degradable structure. FIG. 5G depicts a capture reagent or pull down reagent comprising a plurality of nanoparticle subunits 560 (e.g., organic or inorganic nanoparticles) that are linked by degradable linkers 552 (e.g., nucleic acid restriction sites, photocleavable linkers, etc.). Each nanoparticle subunit 560 comprises a coupling group 558 that is configured to couple with a polypeptide and a coupling group 555 that is configured to be coupled with a solid support or an anchoring group. The capture reagent may be contacted with a plurality of polypeptides 520, where each polypeptide comprises a coupling group 525 that is configured to be coupled with a capture reagent. FIG. 5H depicts the capture reagent or pull down reagent after a degradation process. Each subunit 560 of the capture reagent may be detached from other subunits 560. Each subunit may be further coupled to a polypeptide 520 and comprise a coupling group 555 that is configured to couple with a solid support or an anchoring group.

[0167] A targeting agent, such as a capture reagent or a pull down reagent may be combined with a sample or a composition comprising polypeptides in an amount that constitutes a deficit, at parity, or an excess relative to the total amount of polypeptides present, or the amount of targeted polypeptides present. The amount of a targeting agent contacted or combined with a sample or composition comprising polypeptides may be determined by a level of confidence for the amount of polypeptide present in a sample or polypeptide fraction. For example, the total amount of a polypeptide fraction may be known on a weight basis, but the total number of individual polypeptides within the polypeptide fraction may not be known. Likewise, the total number of individual polypeptides within a polypeptide fraction may be known, but the total number of individual target polypeptides may not be known. A targeting agent may be provided to a targeting separation method in excess to ensure that a sufficient quantity of polypeptide coupling groups are present to capture all targeted polypeptides. When a degradable targeting agent or a capture reagent comprising an anchoring group is utilized in a targeting separation method, an excess of the capture reagent may be utilized to control the display of polypeptides on the surface of a solid support. For example, a degradable capture reagent may degrade into a mixture of polypeptide-coupled subunits and uncoupled subunits. When the mixture of subunits deposits on a solid support, the amount of uncoupled subunits present may control the average spacing between polypeptide-containing sites on the solid support.

[0168] The quantity of a targeting reagent utilized in a targeting separation method may be calculated based upon the total available number of polypeptide-coupling groups on the utilized targeting reagent. A targeting reagent may be provided to a targeting separation method at a particular ratio of polypeptide-coupling groups to available or possibly available polypeptides. A targeting reagent may be provided to a targeting separation method at a particular ratio of polypeptide-coupling groups to available or possibly available polypeptides of about 1:1000, 1:500, 1:100, 1:50, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 50:1, 100:1, 500:1, 1000:1, or more than 1000:1. A targeting reagent may be provided to a targeting separation method at a particular ratio of polypeptide-coupling groups to available or possibly available polypeptides of at least about 1:1000, 1:500, 1:100, 1:50, 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 50:1, 100:1, 500:1, 1000:1, or more than 1000:1. Alternatively or additionally, a targeting reagent may be provided to a targeting separation method at a particular ratio of polypeptide-coupling groups to available or possibly available polypeptides of no more than about 1000:1, 500:1, 100:1, 50:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 1:500, 1:1000, or less than 1:1000.

[0169] A polypeptide fraction prepared by a polypeptide separation method comprising a plurality of sample polypeptides may include polypeptides of a particular size. A polypeptide within a polypeptide fraction may be at least about 0.1 Daltons (Da), 0.5 Da, 1 Da, 5 Da, 10 Da, 50 Da, 100 Da, 200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1 kiloDalton (kDa), 1.5 kDa, 2 kDa, 2.5 kDa, 3 kDa, 3.5 kDa, 4 kDa, 4.5 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1200 kDa, 1400 kDa, 1600 kDa, 1800 kDa, 2000 kDa, 2500 kDa, 3000 kDa, 3500 kDa, 4000 kDa, or more than 4000 kDa. Alternatively or additionally, a polypeptide within a polypeptide fraction may be no more than about 4000 kDa, 3500 kDa, 3000 kDa, 2500 kDa, 2000 kDa, 1800 kDa, 1600 kDa, 1400 kDa, 1200 kDa, 1000 kDa, 900 kDa, 800 kDa, 700 kDa, 600 kDa, 500 kDa, 400 kDa, 300 kDa, 200 kDa, 100 kDa, 90 kDa, 80 kDa, 70 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa, 20 kDa, 15 kDa, 10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5 kDa, 4.5 kDa, 4 kDa, 3.5 kDa, 3 kDa, 2.5 kDa, 2 kDa, 1.5 kDa, 1 kDa, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, 100 Da, 50 Da, 10 Da, 5 Da, 1 Da, 0.5 Da, or less than 0.5 Da.

[0170] A polypeptide within a polypeptide fraction prepared by a polypeptide separation method comprising a plurality of sample polypeptides may contain a minimum or maximum number of amino acid residues. A polypeptide may contain at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000 or more than 40000 amino acid residues. Alternatively or additionally, a polypeptide may contain no more than about 40000, 30000, 20000, 15000, 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 250, 200, 150, 125, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or less than 3 amino acid residues.

[0171] A polypeptide fraction prepared by a polypeptide separation method comprising a plurality of sample polypeptides may comprise a characterized percentage of a total quantity of sample polypeptides in a sample. A percentage of a total quantity of sample polypeptides may be calculated with respect to an appropriate basis, such as compared to an original sample from which the sample polypeptides are derived, as compared to a crude extract prepared from a sample, or as compared to a prior fraction or a prior sample. A polypeptide fraction comprising a plurality of sample polypeptides may contain at least about 0.000001%, 0.000005%, 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, or more than 99.99999% of the total quantity of sample polypeptides on a mass or molar basis. Alternatively or additionally, a polypeptide fraction comprising a plurality of sample polypeptides may contain no more than about 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, 0.0001%, 0.00005%, 0.00001%, 0.000005%, 0.000001%, or less than 0.000001% of the total quantity of sample polypeptides on a mass or molar basis.

[0172] A polypeptide fraction prepared by a polypeptide separation method comprising a plurality of sample polypeptides may have a characterized or characterizable polypeptide diversity after a separation process. A polypeptide diversity of sample polypeptides may be calculated with respect to an appropriate basis, such as compared to an original sample from which the sample polypeptides are derived, as compared to a crude extract prepared from a sample, or as compared to a prior fraction or a prior sample. A polypeptide fraction comprising a plurality of sample polypeptides may have a polypeptide diversity of at least about 0.000001%, 0.000005%, 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, or more than 99.99999% of the total quantity of polypeptide species of the sample from which the plurality of sample polypeptides was derived on a mass or molar basis. Alternatively or additionally, a polypeptide fraction comprising a plurality of sample polypeptides may have a polypeptide diversity of no more than about 99.99999%, 99.9999%, 99.999%, 99.99%, 99.9%, 99.5%, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, 0.0001%, 0.00005%, 0.00001%, 0.000005%, 0.000001%, or less than 0.000001% of the total quantity of polypeptide species of the sample from which the plurality of sample polypeptides was derived on a mass or molar basis.

[0173] A polypeptide fraction may have a characterized or characterizable polypeptide diversity with respect to a known proteome or sub-proteome of an organism from which the sample was derived. A polypeptide diversity of sample polypeptides of a proteome or sub-proteome may be calculated with respect to an appropriate basis, such as compared to an original sample from which the sample polypeptides are derived, as compared to a crude extract prepared from a sample, or as compared to a prior fraction or a prior sample. A polypeptide fraction may have a characterized or characterizable polypeptide diversity of at least about 0.000001%, 0.000005%, 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 150%, 200%, 300%, 400%, 500%, 1000%, or more than 1000% relative to a proteome or sub-proteome on a mass or molar basis. Alternatively or additionally, a polypeptide fraction may have a characterized or characterizable polypeptide diversity of no more than about 1000%, 500%, 400%, 300%, 200%, 150%, 120%, 110%, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005%, 0.0001%, 0.00005%, 0.00001%, 0.000005%, 0.000001%, or less than 0.000001% relative to a proteome or sub-proteome on a mass or molar basis.

[0174] The sample polypeptide content of a polypeptide mixture may be characterized before a polypeptide separation process. The sample polypeptide content of a polypeptide mixture may be characterized as a mass fraction of sample polypeptides relative to total polypeptide content of a polypeptide mixture. For example, a polypeptide mixture containing a mixture of 90% sample polypeptides by weight and 10% internal standard polypeptide by weight may have a characterized sample polypeptide mass fraction of 90% or 0.9. A polypeptide mixture may have a sample polypeptide mass fraction of about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 0.991, 0.992, 0.993, 0.994, 0.995, 0.996, 0.997, 0.998, 0.999, or more than 0.999. A polypeptide mixture may have a sample polypeptide mass fraction of at least about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 0.991, 0.992, 0.993, 0.994, 0.995, 0.996, 0.997, 0.998, 0.999, or more than 0.999. Alternatively or additionally, a polypeptide mixture may have a sample polypeptide mass fraction of no more than about 0.999, 0.998, 0.997, 0.996, 0.995, 0.994, 0.993, 0.992, 0.991, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, 0.9, 0.89, 0.88, 0.87, 0.86, 0.85, 0.84, 0.83, 0.82, 0.81, 0.8, 0.75, 0.7, 0.65, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, 0.01 or less than 0.01.

[0175] A polypeptide separation process may occur in a medium that facilitates the separation process. A polypeptide mixture solvent composition may comprise a fluid medium, such as an aqueous solvent. In some cases, a polypeptide separation process may occur in a polypeptide mixture separation composition that is in contact with the solid support. A polypeptide mixture solvent composition for storage of a sample or partially purified sample may be utilized for a polypeptide separation process. A polypeptide mixture solvent composition may comprise one or more polypeptide composites in solution or suspension. A polypeptide mixture solvent composition may be formulated to be a homogeneous liquid medium. A polypeptide mixture solvent composition may be formulated to be a single-phase liquid medium. A polypeptide mixture solvent composition may be formulated to be a multi-phase liquid medium, such as an oil-in-water emulsion or a water-in-oil emulsion. For a polypeptide mixture solvent composition formulated as an emulsion, anchoring groups or polypeptide composites may be solvated or suspended within the dissolved phase.Polypeptide Functionalization

[0176] Polypeptides (e.g. sample polypeptides or standard polypeptides) or a polypeptide fraction may be treated with one or more modifying agents to add functional groups to some or all polypeptides within a sample or polypeptide fraction. The methods and compositions set forth below may generally be exemplified with reference to a modifying agent, functional group, polypeptide and / or a protein; however, it will be understood that the examples can be extended to populations of modifying agents, functional groups, polypeptides, and / or proteins. For example, a population having the same species of protein can be used with a population of the same species of modifying agent and / or functional group, a population having different species of protein can be used with a population of the same species of modifying agent and / or functional group, a population having the same species of protein can be used with a population of different species of modifying agent and / or functional group, or a population having different species of protein can be used with a population of different species of modifying agent and / or functional group. Moreover, methods and compositions may be exemplified below with reference to proteins; however, it will be understood that the examples can be extended to other polypeptides such as sample polypeptides or standard polypeptides.

[0177] A functional group may be added to a polypeptide from a polypeptide fraction for any of a variety of purposes, including altering chemical properties (e.g., altering electrical charge, altering hydrophobicity, altering reactivity), adding groups that improve detectability of polypeptides (e.g., fluorophores, luminescent particles, radiolabels), modulating activity (e.g. modification of amino acids at or near the active site of an enzyme, or binding to an inhibitor or activator of the enzyme) or adding groups that alter the separability of polypeptides (e.g., magnetic particles, hydrophobic particles, charged particles, linkers for attachment to solid-phase materials etc.).

[0178] A polypeptide fraction may be treated with one or more modifying agents to add a reactive functional group to some or all polypeptides within a polypeptide fraction. In some cases, a polypeptide fraction may be treated with one or more modifying agents to add a functional group to some or all polypeptides that are configured to interact with a second functional group. A polypeptide with a functional group may interact with a second molecule with a second functional group to form a polypeptide composite. A polypeptide composite may be formed for any of a variety of purposes, including altering the polypeptide's chemical properties (e.g., altering electrical charge, altering hydrophobicity, altering reactivity), altering the polypeptide's activity (e.g. inhibiting or activating enzymatic activity), adding groups that improve detectability of the polypeptide (e.g., fluorophores, luminescent particles, radiolabels), or adding groups that alter the separability of the polypeptide (e.g., magnetic particles, hydrophobic particles, charged particles).

[0179] A polypeptide from a sample or a polypeptide fraction may be functionalized with one or more functional groups. A functional group may be added to a polypeptide at a terminal amino acid residue, for example at the terminal carboxyl moiety or side chain moiety of the C-terminal amino acid or at the terminal amino moiety or side chain moiety of the N-terminal amino acid. A functional group may be added to a polypeptide at the side chain of any given non-terminal amino acid within the polypeptide. A functional group may be added to a polypeptide at any amino acid containing an amine or thiol side chain. A functional group may be added to a cysteine, asparagine, glutamine, lysine, histidine, tryptophan, or arginine amino acid residue. A polypeptide may be functionalized at more than one amino acid (e.g., all or nearly all amine side chains may be functionalized for a polypeptide). A polypeptide may have a functional group added to one or more type of side chain. For example, some or all side chains having amines may be functionalized, some or all side chains having carbonyls may be functionalized, some or all side chains having phosphates may be functionalized, some or all side chains having hydroxyls may be functionalized, or some or all side chains having thiols may be functionalized. Functionalization can be carried out to add the same functional group to multiple side chains of the polypeptide and the multiple side chains can optionally be the same or different type of side chain. A polypeptide may be functionalized to add more than one type of functional group to the polypeptide (e.g., adding azides to all amines and NHS esters to all thiols) and different types of functional groups can be added to the same type of side chain. Alternatively, different types of functional groups can be added to different types of side chains respectively. A polypeptide may be functionalized with more than one type of functional group with all functional groups added for the same purpose (e.g., increasing reactivity). A polypeptide may be functionalized with more than one type of functional group with differing functional groups added for differing purposes (e.g., increasing reactivity and altering hydrophobicity).

[0180] A polypeptide may be functionalized at any stage of a sample preparation process set forth herein or known in the art. A polypeptide may be functionalized before it has been separated, isolated or extracted from a sample. A polypeptide may be functionalized while it is still contained within a sample. A polypeptide may be functionalized during the processing of a sample but before any polypeptides have been separated from the sample (e.g., after cell lysis). A polypeptide may be functionalized during or after separation, isolation, or extraction from a sample. A functionalization process may be repeated one or more times to ensure complete or near-complete functionalization of all polypeptides derived from a sample.

[0181] FIG. 6A depicts a scheme for preparing polypeptides that are modified with a functional group in an intact biological sample. In this scheme, the polypeptides are modified with a functional group before they are extracted from the sample to yield a functionalized polypeptide fraction. A biological material 600 (e.g., a cell) may be contacted with a reagent 660 that is configured to functionalize a polypeptide. The reagent 660 may be provided with one or more additional reagents that are necessary to induce a reaction between the reagent 660 and any available polypeptides. The reagent 660 may be contacted with the biological material in the presence of a detergent. The reagent 660 may be contacted with the biological material in the presence of a standard polypeptide such as a reagent standard polypeptide. The biological material 600 may include any biological component, including but not limited to cell walls, cell membranes 670, nuclear membranes 672, organelles 674, genetic materials 650 (e.g., chromosomes, plasmids), fibrils or other structural components, capsids, cartilage, bone, exoskeleton, exocellular proteins, cytoplasmic proteins 610, nuclear proteins 620, organelle proteins 630, and membrane-bound or membrane-linked proteins 640. The reagent 660 may react with any available polypeptides in sample to form functionalized polypeptides, such as functionalized cytoplasmic proteins 611, functionalized nuclear proteins 621, functionalized organelle proteins 631, and functionalized membrane proteins 641. The reagent 660 may incidentally functionalize other non-polypeptide components of the sample. After the sample has been functionalized with the reagent 660, the sample may be processed as described above to separate, isolate, or extract the polypeptides from the sample, producing a polypeptide fraction including the functionalized polypeptides, and a waste fraction composing other components such as the genetic materials 650 and fragmented cellular debris 680 (e.g., membrane fragments, organelle fragments). The polypeptide fraction may be separated, isolated, or extracted from the non-polypeptide fraction in the presence of a detergent. The polypeptide fraction may be separated, isolated, or extracted from the non-polypeptide fraction in the presence of a standard polypeptide, such as a separation standard polypeptide, isolation standard polypeptide, or extraction standard polypeptide.

[0182] FIG. 6B depicts a scheme for preparing polypeptides that are modified with a functional group, the polypeptides being from an intact biological sample. In this scheme, the polypeptides are modified with a functional group after they are extracted from the sample to yield a functionalized polypeptide fraction. A biological material 600 (e.g., a cell) may be processed as described herein to release polypeptides contained within the biological material 600. The biological material 600 may include any biological component, including but not limited to cell walls, cell membranes 670, nuclear membranes 672, organelles 674, genetic materials 650 (e.g., chromosomes, plasmids), fibrils or other structural components, capsids, cartilage, bone, exoskeleton, exocellular proteins, cytoplasmic proteins 610, nuclear proteins 620, organelle proteins 630, and membrane-bound or membrane-linked proteins 640. After the biological material 600 has been processed to release polypeptides, the polypeptides may be contacted with a reagent 660 that is configured to functionalize a polypeptide. The reagent 660 may be provided with one or more additional reagents that are necessary to induce a reaction between the reagent 660 and any available polypeptides. The reagent 660 may functionalize the available polypeptides to produce functionalized polypeptides, such as functionalized cytoplasmic proteins 611, functionalized nuclear proteins 621, functionalized organelle proteins 631, and functionalized membrane proteins 641. Functionalized polypeptides may be extracted, isolated, or separated from the non-polypeptide components (e.g., genetic material 650, cellular debris 680 ) to yield a polypeptide fraction. The reagent 660 may be introduced before or after the polypeptides have been separated from the residual non-polypeptide components.

[0183] FIG. 7A depicts a scheme for preparing polypeptides that are modified with a functional group, the sample polypeptides being from a sample and in solution phase (e.g., blood plasma, water samples). The sample may comprise polypeptides 710 suspended or solvated within a liquid medium, as well as other components 700 (e.g., cells, viral particles, non-biological particles) that are not of interest in a polypeptide assay. The polypeptides 710 may be separated from the other components before the polypeptides 710 are functionalized. Methods such as precipitation, centrifugation, filtration, liquid-phase extraction, solid-phase extraction, size-exclusion chromatography, and liquid chromatography, or a combination thereof, may be utilized to separate, isolate, or extract the other components 700 from the solution phase polypeptides 710. After the solution phase polypeptides 710 have been collected in a polypeptide fraction, the polypeptides may be contacted with a functionalizing reagent 720 that is configured to add a functional group to the polypeptide. The functionalizing reagent 720 may be provided with one or more additional reagents that are necessary to induce a reaction between the reagent 720 and any available polypeptides. The functionalizing reagent 720 may functionalize the available polypeptides to produce a functionalized polypeptide fraction 740. The polypeptide fraction may undergo additional purification and / or preparation processes before or after the functionalization step.

[0184] FIG. 7B depicts an alternative scheme for preparing a polypeptide fraction comprising polypeptides that are modified with a functional group from a sample where the polypeptides of interest are in solution phase (e.g., blood plasma). The sample may comprise polypeptides 710 suspended or solvated within a liquid medium, as well as other components 700 (e.g., cells, viral particles) that are not of interest in a polypeptide assay. The sample may be contacted with a functionalizing reagent 720 before the polypeptides 710 of interest have been separated from any other components 700. The functionalizing reagent 720 may be provided with one or more additional reagents that are necessary to induce a reaction between the reagent 720 and any available polypeptides. The functionalizing reagent 720 may functionalize solution phase polypeptides 710 as well as other components 700 to produce functionalized polypeptides 711 mixed with functionalized biological materials 701. After the functionalization step, the functionalized polypeptides 711 may be separated, isolated, or extracted from the other functionalized biological materials 701, thereby yielding a functionalized polypeptide fraction 740.

[0185] FIG. 8A depicts a scheme for preparing a polypeptide fraction comprising polypeptides that are modified with a functional group when the sample is to be extracted from a curated solid medium 800 (e.g., rock, a filter). The solid medium 800 may include adhering polypeptides 810 or adhering non-polypeptide components 820 (e.g., mineral particles). The solid medium 800 may be subjected to one or more washing or extraction processes that separate the polypeptides 810 from the solid medium 800 and any non-polypeptide components 820. The separated polypeptides 810 may be contacted with a functionalizing reagent 830 to produce a functionalized polypeptide fraction 811. The functionalizing reagent 830 may be provided with one or more additional reagents that are necessary to induce a reaction between the reagent 830 and any available polypeptides.

[0186] FIG. 8B depicts an alternative scheme for preparing a polypeptide fraction comprising polypeptides that are modified with a functional group when the sample is to be extracted from a curated solid medium 800 (e.g., rock, a filter). The solid medium 800 may include adhering polypeptides 810 or adhering non-polypeptide components 820 (e.g., mineral particles). The entire sample may be contacted with a functionalizing reagent 830. The functionalizing reagent 830 may be provided with one or more additional reagents that are necessary to induce a reaction between the reagent 830 and any available adhering polypeptides 810 to produce a functionalized polypeptide fraction 811. The solid medium 800 may be subjected to one or more washing or extraction processes that separate the functionalized polypeptide fraction 811 from the solid medium 800 and any non-polypeptide components 820.

[0187] Standard polypeptides can be particularly useful in methods for preparing polypeptides modified with functional groups, such as those exemplified above in reference to FIGs 6 - 8. One or more different standard polypeptides, such as species of standard polypeptide set forth elsewhere herein, can be present during at least one of the steps in each scheme. One or more different standard polypeptides can be added before, during or after any of the steps, for example, to provide a means to characterize the step or subsequent step(s). Standard polypeptides present during a particular step can optionally be used to characterize a product of the step. Exemplary characterizations include, but are not limited to quantifying sample polypeptides or other product(s) of a method or step exemplified herein, identifying a property of a sample polypeptide or other product of a method or step exemplified herein, or determining the quality of a sample polypeptide or other product of a method or step exemplified herein. The standard polypeptides can optionally be functionalized as exemplified in the methods above, for example, the standard polypeptides being treated simultaneously with, and in the same sample as, sample polypeptides. A standard polypeptide can have a known or predicted reactivity toward one or more reagent that is used in a functionalization reaction. In some cases a plurality of different standard polypeptides can be present in a functionalization reaction, wherein each of the different standard polypeptides has a different reactivity toward one or more reagents used in the functionalization reaction. As such, one or more standard polypeptides can be used as functionalization standards.

[0188] Polypeptides may be conjugated with a functional group for any of a variety of purposes. A functional group may be added to a polypeptide to increase or alter the reactivity of the polypeptide. In some cases, a polypeptide may be functionalized with a functional group that is configured to react with another chemical species by a "click" reaction (see, for example, U.S. Pat. Nos. 6,737,236 and 7,427,678 ) such as azide alkyne Huisgen cycloaddition reactions, which use a copper catalyst (see, for example, U.S. Pat. Nos. 7,375,234 and 7,763,736 ); or Copper-free Huisgen reactions ("metal-free click") using strained alkynes or triazine-hydrazine moieties which can link to aldehyde moieties (see, for example, U.S. Pat. No. 7,259,258

[0189] ). A polypeptide may be functionalized with a functional group to facilitate coupling or conjugation of the polypeptide to other molecules or materials. For example, a polypeptide may be coupled or conjugated to a complementary species such as a biomolecule, fluorescent label, or a surface. The polypeptide may be coupled or conjugated to the complementary species by a covalent or non-covalent interaction of the functional group on the polypeptide with a complementary functional group, chemical species or active site on the complementary species. Functional groups for coupling or conjugation of a polypeptide to a complementary species may comprise a single-stranded nucleic acid, a double-stranded nucleic acid, a nucleophile, an electrophile, a coordination compound (e.g., a silane), or a moiety capable of forming hydrogen bonds (e.g., alcohols, carboxylic acids, ketones).

[0190] The extent of functionalization of a functionalized polypeptide fraction may be characterized or quantified. The extent of functionalization may include one or both of two aspects: 1) the quantity of polypeptides within a polypeptide fraction that receive at least one functionalization; and 2) the average number of functionalizations per polypeptide within a polypeptide fraction. A polypeptide fraction may be functionalized to ensure that all polypeptides contain at least one added functional group. A polypeptide fraction may be functionalized until there is an average of at least one functional group per polypeptide. In some cases, an individual polypeptide from a polypeptide fraction may have no added functional groups. In some cases, an individual polypeptide from a polypeptide fraction may have more than one added functional group. In some cases, a polypeptide may have a functional group on every available target for functionalization (e.g., all side chain amines are functionalized with an azide). The extent of functionalization for individual molecules within a polypeptide fraction may follow some quantifiable distribution, such as a Poisson distribution, binomial distribution, beta-binomial distribution, hypergeometric distribution, or bimodal distribution. The extent of functionalization may be specific to a single type of functional group if more than one functional group is added to a polypeptide fraction. The extent of functionalization may be a global measure of functionalization if more than one functional group is added to a polypeptide fraction. The extent of functionalization for a functionalized polypeptide fraction may be measured by any suitable method. For example, quantity of a polypeptide fraction with at least one functionalization may be assessed by conjugating a macromolecule (e.g., PEG) to functionalized polypeptides, then separating by a method such as size-exclusion chromatography to quantify the relative amounts of functionalized and non-functionalized polypeptide in a polypeptide sample. Likewise, functionalization of a polypeptide may cause a change in property (e.g., hydrophobicity, polarity) that may permit chromatographic separation on the basis of the property (e.g., hydrophobic exchange chromatography, reverse-phase liquid chromatography). In another example, the average number of functionalizations per molecule may be determined by conjugation of labeled dyes to the added functional groups, thereby permitting quantitation of total dye conjugated by a colorimetric or fluorescent assay. Functionalization of polypeptides may be directly measured by an analytical technique such as mass spectrometry (MS), or spectroscopy (e.g., infrared, UV-vis, Raman, etc.).

[0191] A plurality of functionalized polypeptide may be coupled to a plurality of molecules to determine an extent of functionalization for the plurality of polypeptides. The plurality of functionalized polypeptides may be coupled to macromolecules such as biopolymers ( e.g., polypeptides, nucleic acids, polysaccharides). Macromolecules may be coupled or conjugated to polypeptides by a cleavable or reversible linker (e.g., a photocleavable linker). Macromolecules may be larger than a polypeptide (e.g., by weight or radius) or chemically dissimilar relative to a functionalized polypeptide (e.g., by hydrophobicity polarity, etc.) to permit fractionation of polypeptides by number of macromolecules incorporated. For example, size-exclusion chromatography may be utilized to produce fractions of polypeptides coupled to 1 macromolecule, 2 macromolecules, 3 macromolecules, etc. Relative quantities of recovered fractions may be correlated to the relative numbers of functionalizations per polypeptide molecule. In some cases, the estimated number of functionalizations per polypeptide may comprise a size proxy (e.g., larger polypeptides may contain more amino acid residues that are functionalized). In some cases, each size-based polypeptide fraction may be individually coupled to differing anchoring groups to provide identifiable fractions based upon polypeptide size, approximate number of functionalized amino acids, or approximate solvent accessible area of each polypeptide fraction.

[0192] A suitable functionalized polypeptide fraction for a polypeptide assay may have a quantified extent of functionalization that meets a threshold value. The threshold value (e.g., a maximum or minimum threshold value) may represent the extent of functionalization needed to obtain an accurate characterization of polypeptide fraction. The threshold value for extent of functionalization may depend upon the polypeptide assay to be performed. For example, a qualitative assay (e.g., presence or absence of a biomarker) may require a lower threshold for extent of functionalization to ensure detection of a polypeptide. In another example, a quantitative assay (e.g., polypeptide sizing) may have an upper threshold for extent of functionalization if an excess of functionalization may exceed a detection limit of a measurement assay. In some cases, the extent of functionalization may be quantified as the average number of functionalizations per polypeptide molecule or per mass of polypeptide. In some cases, the extent of functionalization may be quantified as the percentage of polypeptides to receive at least one functional group. The threshold average number of functionalizations per polypeptide molecule may be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50 or greater than 50 functionalizations per molecule. Alternatively or additionally, the average number of functionalizations per polypeptide molecule may be no more than about 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less than 0.1 functionalizations per molecule. The threshold percentage of functionalized polypeptide in a polypeptide fraction may be at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or greater than 99.9% of the total polypeptide in the fraction measured by mass or molarity. Alternatively or additionally, the threshold percentage of functionalized polypeptide in a polypeptide fraction may be no more than about 99.9%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less than 1% of the total polypeptide in the fraction measured by mass or molarity.

[0193] The extent of functionalization for one or more sample polypeptides may be quantified relative to the extent of functionalization for one or more standard polypeptides, such as standard polypeptide(s) having a known or predicted reactivity with one or more reagents used in the functionalization reaction. Accordingly, a threshold value for functionalization of one or more sample polypeptide may be quantified relative to functionalization of one or more standard polypeptide. Moreover, properties of functionalized sample polypeptides may be characterized relative to known or expected properties of functionalized standard polypeptides.

[0194] In some cases, a functionalization standard may include one or more polypeptides that are configured to provide a quantitative measure of polypeptide functionalization. For example, a functionalization standard may comprise a polypeptide that contains an amino acid abundance or overabundance (e.g., relative to a natural proteomic amino acid abundance) of an amino acid residue that is functionalized (e.g., a 200 residue polypeptide comprising 100 lysine residues). Such a polypeptide may provide a more sensitive measure for the extent of functionalization. In another example, a functionalization standard may comprise a polypeptide comprising an amino acid abundance or overabundance (e.g., relative to a natural proteomic amino acid abundance) of an amino acid residue that is off target for a functionalization chemistry (e.g., a 200 residue polypeptide comprising 100 arginine residues, for a lysine-targeting functionalization chemistry). In another example, a functionalization standard may comprise a polypeptides comprising functionalization target residues (e.g., lysine, cysteine, etc.) in differing sequence contexts. For example, a functionalization standard may comprise a polypeptide comprising lysine residues that are buried in a tertiary structure to assess the extent of functionalization of lysines based upon accessibility. In some cases, a functionalization standard may comprise a plurality of polypeptides that are configured to determine an effect of functionalization of a polypeptide on affinity agent binding to a polypeptide epitope. For example, a functionalization polypeptide standard may comprise a plurality of polypeptides, in which each polypeptide of the plurality of polypeptides comprises a same epitope, and in which each polypeptide of the plurality of polypeptides comprises a functionalized amino acid residues (e.g., a functionalized lysine, etc.) with a known position relative to the epitope (e.g., within at least 1, 2, 3, 4, 5, or more than 5 amino acid residues of the epitope). Such a functionalization standard may provide a quantitative or qualitative measure of the effect of the functionalized amino acid on affinity agent binding to the epitope.

[0195] Functionalization reactions may be repeated for a particular polypeptide fraction. A functionalization reaction may be repeated for a particular polypeptide fraction to obtain a larger extent of functionalization. A functionalization reaction may be repeated under differing conditions to functionalize polypeptides that are resistant to functionalization under the initial reaction conditions. Alternatively, a functionalization reaction may be performed only once or for a limited amount of time to prevent excessive functionalization of a plurality of polypeptides. For example, functionalization can be carried out under differing denaturation conditions to allow increased access to side chain moieties that are differentially accessible under those conditions. For example, functionalization can be carried out at different temperatures (e.g. temperatures higher than the physiological range for the sample from which the polypeptides are derived), different pH (e.g. pH higher or lower than the physiological range for the sample from which the polypeptides are derived), different ionic strengths (e.g. ionic strength lower or higher than the physiological range for the sample from which the polypeptides are derived), different polarity (e.g. polarity that is higher or lower than the physiological range for the sample from which the polypeptides are derived), different species of chemical denaturant and / or different concentrations of chemical denaturant. A functionalization reaction may be repeated using a differing reagent that more effectively targets any residues not functionalized during the first functionalization reaction. In some cases, additional reagent may be supplied to a functionalization reaction during the course of the reaction to replenish depleted reagents, shift a chemical equilibrium, or alter a reaction rate. In some cases, a plurality of sample polypeptides may be functionalized then separated to isolate functionalized polypeptides from unfunctionalized polypeptides. In such cases, the unfunctionalized polypeptide fraction may be functionalized a second time to ensure near-complete or complete functionalization of all sample polypeptides.

[0196] In some cases, functionalization may be carried out with co-solvents or alternative solvents. A functionalization solvent may be aqueous or non-aqueous. A solvent or co-solvent may be an organic solvent or an anhydrous solvent. Solvent choice may be influenced by the target for functionalization and the specificity of the reaction between the polypeptide and the functionalization compound. For example, altering a solvent from DMSO to aqueous PBS buffer may shift the selectivity of a dinitroimidazole reaction with a thiol-containing sidechain from lysine to cysteine. A functionalization solvent may also comprise a catalyst, such as a dissolved catalyst or a solid catalyst, that promotes an increased rate or specificity of polypeptide functionalization. For example, the reaction of an amine-containing polypeptide with a proton-acceptor catalyst in an anhydrous solvent (e.g., diisopropylethylamine (DiPEA), triethylamine (TEA), N-methyl morpholine (NMM)) may favor reaction with the amine over hydrolysis. A functionalization reaction may occur in the presence of an enzyme that is configured to modify an amino acid side chain or add a functional group to an amino acid side chain or terminal residue.

[0197] A functionalization reaction may be chosen for a polypeptide fraction based upon the speed of the reaction. Selection of functionalization reactions may be based upon various factors, including the stability of the sample, the time available to prepare the sample, the known or expected chemical composition of the sample, the available functionalizing reagents, and the type of characterization assay to be performed. For example, a time-sensitive sample may be functionalized with an enzymatic or catalytic approach (e.g., sterically-hindered bases for amine side chains), or in the presence of a high concentration of the functionalization reagent. In another example, a sample may be shelf stable for up to a day at room temperature. This sample may be functionalized with an anhydrous functionalization chemistry such as carbene reactions (slower due to the steps of rendering a polypeptide sample anhydrous). A functionalization reaction may occur in the presence of a surfactant and / or a detergent. A functionalization reaction may occur in the presence of a co-solvent that is configured to improve the rate, conversion or selectivity of a functionalization reaction. For example, an aqueous functionalization medium may be modified to include a miscible co-solvent (e.g., ethanol, methanol, acetone) or an immiscible co-solvent (e.g., dimethyl ether, carbon tetrachloride) during a functionalization reaction.

[0198] Functional groups capable of rapidly forming covalent bonds with other chemical species may be of particular interest for the functionalization of polypeptides. In general, functional groups of interest will include most common species for bioconjugation. Such functional groups may include "click" reagents that are capable of forming highly specific products with complementary functional groups in a rapid and irreversible fashion.

[0199] A functionalization reaction of a polypeptide may be performed in a fashion that ensures that some or all reactive sites in a polypeptide become functionalized. For example, an amine-functionalizing chemistry (e.g., NHS-azide conjugation) may be performed with an excess of the azide-containing compound to ensure complete reaction of all amine side chains. A functionalization reaction may be performed with a functional group:polypeptide molar ratio of about 1:1000, 1:500, 1:250, 1:100, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 25:1, 30:1, 50:1, 100:1, 250:1, 500:1, 1000:1, or more. A functionalization reaction may be performed with a functional group:polypeptide molar ratio of at least about 1:1000, 1:500, 1:250, 1:100, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 25:1, 30:1, 50:1, 100:1, 250:1, 500:1, 1000:1, or more. Alternatively or additionally, a functionalization reaction may be performed with a functional group:polypeptide molar ratio of no more than about 1000:1, 500:1, 250:1, 100:1, 50:1, 30:1, 25:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500, 1:1000, or more.

[0200] A functionalization reaction may occur at a fixed temperature. In some cases, a functionalization reaction may occur at a first temperature for a fixed amount of time, then a second temperature for a fixed amount of time. A functionalization reaction between a polypeptide and a reactive group may occur at a temperature of about - 80 °C, - 70 °C, - 60 °C, - 50 °C, - 40 °C, - 30 °C, - 20 °C, - 10 °C, - 5° C, 0 °C, 4 °C, 10 °C, 20°C, 30°C, 37 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, or about 95 °C. A functionalization reaction between a polypeptide and a reactive group may occur at a temperature of at least about - 80 °C, - 70 °C, - 60 °C, - 50 °C, - 40 °C, - 30 °C, - 20 °C, - 10 °C, - 5 °C, 0 °C, 4 °C, 10 °C, 20 °C, 30°C, 37 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, or at least about 95 °C. Alternatively or additionally, a functionalization reaction between a polypeptide and a reactive group may occur at a temperature of no more than about 95 °C, 90 °C, 80 °C, 70 °C, 60 °C, 50 °C, 40 °C, 37 °C, 30 °C, 20 °C, 10°C, 4°C, 0 °C, - 5 °C, - 10°C, - 20 °C, - 30 °C, - 40 °C, - 50 °C, -60°C, - 70 °C, or about - 80 °C.

[0201] A functionalization reaction between a polypeptide and a reactive group may occur for a fixed amount of time. A functionalization reaction between a polypeptide and a reactive group may occur for about 1 min, 30 mins, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 wks, 3 wks, 4 wks, 1 mth, or more than 1 mth. A functionalization reaction between a polypeptide and a reactive group may occur for at least about 1 min, 30 mins, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 wks, 3 wks, 4 wks, 1 mth, or more than 1 mth. Alternatively or additionally, a functionalization reaction between a polypeptide and a reactive group may occur for no more than about 1 mth, 4 wks, 3 wks, 2 wks, 10 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 23 hrs, 22 hrs, 21 hrs, 20 hrs, 19 hrs, 18 hrs, 17 hrs, 16 hrs, 15 hrs, 14 hrs, 13 hrs, 12 hrs, 11 hrs, 10 hrs, 9 hrs, 8 hrs, 7 hrs, 6 hrs, 5 hrs, 4 hrs, 3 hrs, 2 hrs, 1 hr, 30 mins, 1 min, or less than 1 min.

[0202] In some cases, chemical conjugation techniques may be applied for creating biomaterial-biomolecule conjugates. Functional groups used for bioconjugation may be native to the biomolecule or may be incorporated synthetically. In the illustrations below, R and R' may be a biomolecule (for example, but not limited to: SNAPs, polypeptides, nucleic acids, carbohydrates, lipids, metabolites, small molecules, monomers, oligomers, polymers) and / or a solid support.

[0203] A polypeptide or particle can be modified to incorporate a reactive moiety that will in turn participate in a subsequent attachment reaction, such as a reaction for attaching a particle to a polypeptide, a reaction for attaching a particle to a solid support or a reaction for attaching a polypeptide to a solid support. Functionalizing one or both of such reaction partners may improve the efficiency or speed of subsequent attachment between the partners. For example, a sulfhydryl group (-SH) or amine (-NH 2 ) of a polypeptide or particle may be functionalized to allow for greater reactivity or efficiency of an attachment reaction. Exemplary functionalization chemistries are set forth below. For example, a reaction set forth below can be used to attach a reactive moiety to a particle, and R and R' may represent the particle or the reactive moiety. In other cases, a reaction set forth below can be used to attach a reactive moiety to a polypeptide, and R and R' may represent the polypeptide or the reactive moiety. Similarly, a reaction set forth below can be used to attach a reactive moiety to a solid support and, R and R' may represent the solid support or the reactive moiety. It will be understood that the reactions set forth below can be used to attach a particle to a polypeptide and R and R' may represent the polypeptide or the particle.

[0204] Amine moieties can be functionalized to attach a reactive moiety or other moiety. In some cases, an isothiocyanate moiety may react with nucleophiles such as amines, sulfhydryls, the phenolate ion of tyrosine side chains or other molecules to form an isothiourea linkage.

[0205] An isocyanate moiety can react with an amine to form a stable isourea linkage.

[0206] An acyl azide can react with a primary amine to form an amide linkage.

[0207] An N-hydroxysuccinimide (NHS) ester can react with an amine to form an amide linkage.

[0208] A sulfonyl chloride can react with a primary amine to form a sulfonamide linkage.

[0209] Carbonyl moieties such as aldehydes, ketones, and glyoxals can react with amines to form Schiff base intermediates and the addition of sodium borohydride or sodium cyanoborohydride can reduce the Schiff base intermediate to form a secondary amine linkage.

[0210] An epoxide or oxirane can react with a primary amine, sulfhydryl, or hydroxyl to create a secondary amine, thioether, or ether linkage, respectively.

[0211] A carbonate can react with nucleophiles such as amines to form a carbamate linkage.

[0212] An aryl halide, such as fluorobenzene derivative, can react with an amine to form an arylamine linkage. Other nucleophiles such as thiol, imidazolyl, and phenolate groups can also react with an aryl halide to form a stable linkage.

[0213] An amine can react with an imidoester to form an amidine linkage.

[0214] A carbodiimide can be used as a zero-length crosslinking agents to mediate formation of an amide or phosphoramidate linkage between a carboxylate group and an amine, or between a phosphate and an amine, respectively. Carbodiimides are zero-length reagents because in forming these bonds no additional chemical structure is introduced between the conjugating molecules. A carbodiimide can be used to activate a phosphate to an intermediate phosphate ester that in turn reacts with an amine to form a phosphoramidate linkage.

[0215] Squarates and cyanomethyl acyl sulfonamides, such as those set forth in Abbasov et al., Nature Chemistry 13: 1081-1092 (2021), can be used to modify amino moieties on lysines.

[0216] In some cases, a thiol may be functionalized or modified. For example, the thiol group of cysteine is the most nucleophilic functional group found among the 20 polypeptideogenic amino acids. Through careful control of pH, selective modification over other nucleophilic amino acid residues such as lysine can be readily achieved. Moreover, thiol modification of oligonucleotides may be used to enable derivatization. In some cases, the unique nucleophilicity of thiols can be exploited for selective reaction with a number of alternative electrophiles, which allow efficient and selective attachment to be achieved. For example, one such group includes α-halocarbonyls, with iodoacetamide based reagents finding particular utility. Higher thiol selectivity may be achieved using less electrophilic bromo- and even chloro-derivatives, though reactivity is also reduced. Methylsulfonyl heteroaromatic derivatives can also be used for thiol-specific conjugation. In other cases, alternative thiol-functional groups, such as disulfide-bridging pyridazinediones, carbonylacrylic reagents, and cyclopropenyl ketones may be utilized for bioconjugation.

[0217] Three forms of activated halogen derivatives that can be used for functionalization of sulfhydryls include haloacetyl, benzyl halides, and alkyl halides. In each of these compounds, the halogen group may be easily displaced by an attacking nucleophilic substance to form an alkylated derivative with loss of HX (where X is the halogen and the hydrogen comes from the nucleophile).

[0218] The double bond of a maleimide can undergo an alkylation reaction with a sulfhydryl to form a thioether linkage.

[0219] A sulfhydryl can react with an aziridine to form a thioether bond.

[0220] An acryloyl can be reacted with a sulfhydryl to create a thioether linkage.

[0221] Although aryl halides are commonly used to modify amine-containing molecules to form aryl amine derivatives, they also may react quite readily with sulfhydryl groups. For example, a fluorobenzene can react with a sufhydryl to form an aryl thioether linkage. Conjugates formed with sulfhydryl groups are reversible by cleaving with an excess of thiol (such as DTT).

[0222] A vinyl sulfone can be reacted with a sulfhydryl to form a beta-thiosulfonyl linkage.

[0223] Compounds containing a disulfide group can participate in a disulfide exchange reaction with another thiol.

[0224] For example, a pyridyl dithiol can undergo an interchange reaction with a free sulfhydryl to yield a single mixed disulfide product.

[0225] In another example, sulfhydryl groups activated with the leaving group 5-thio-2-nitrobenzoic acid can be used to couple free thiols by disulfide interchange. The disulfide of Ellman's reagent can undergo disulfide exchange with a free sulfhydryl to form a mixed disulfide with concomitant release of one molecule of the chromogenic substance 5-sulfido-2-nitroben-zoate, also called 5-thio-2-nitrobenzoic acid (TNB). The TNB-thiol group can again undergo interchange with a sulfhydryl to yield a disulfide crosslink. Upon coupling with a sulfhydryl compound, the TNB group is released.

[0226] In some cases, disulfide reduction may be performed using thiol-containing compounds such as TCEP, DTT, 2-mercaptoethanol, or 2-mercaptoethylamine. Dinitroimidazoles, such as 1,4-dinitorimidazoles can react with cysteine under acidic to neutral conditions to form a (4-nitroimidazole)-thiol product. See, for example, Luo et al., Nat. Comm. 10: 142 (2019).

[0227] Optionally, a carboxylate may be utilized for functionalization or modification of a polypeptide, particle or other substance. For example, N,N'-Carbonyl diimidazole (CDI) can react with carboxylic acids under nonaqueous conditions to form N-acylimidazoles of high reactivity. An active carboxylate can then react with amines to form amide bonds or with hydroxyl groups to form ester linkages.

[0228] In some cases, carbodiimides function as zero-length crosslinking agents capable of activating a carboxylate group for coupling with an amine-containing compound for attachment. Carbodiimides can be used to mediate the formation of amide or phosphoramidate linkages between a carboxylate and an amine or a phosphate and an amine. N,N'-Disuccinimidyl carbonate (DSC) is highly reactive toward nucleophiles. In aqueous solutions, DSC can be hydrolyzed to form two molecules of N-hydroxysuccinimide (NHS) with release of one molecule of CO 2 . In nonaqueous environments, the reagent can be used to activate a hydroxyl group to a succinimidyl carbonate derivative. DSC-activated hydroxylic compounds can be used to conjugate with amine-containing molecules to form stable crosslinked products.

[0229] In some cases, sodium periodate can be used to oxidize hydroxyl groups on adjacent carbon atoms, forming reactive aldehyde moieties suitable for coupling with amine- or hydrazide-containing molecules for conjugation. Optionally, reactive alkyl halogen compounds can be used to specifically modify hydroxyl groups for attachment.

[0230] In some cases, modification reagents can add a receptor or ligand moiety to a polypeptide, particle or other substance set forth herein. For example, amines, carboxylates, sulfhydryls, carbohydrate groups and other reactive sites can be functionalized with a biotin or (strept)avidin moiety. In some cases, photoreactive biotinylation reagents are used to add a biotin group to a particle or polypeptide, for example, when not containing convenient functional groups for modification. Alternatively, a linkage set forth above can be used. For example, carboxylate-containing biotin compounds can be coupled to amines via a carbodiimide-mediated reaction using EDC. In some cases, NHS-iminobiotin can be used to label amine-containing molecules with an iminobiotin moiety. In some cases, Sulfo-NHS-SS-biotin (also known as NHS-SS-biotin) can be used to modify amine-containing polypeptides, paricles or other substances. In some cases, 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexane-carboxamido]butane reacts with sulfhydryls to form stable thioether linkages. In some cases, N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide, where the reagent contains a 1,6-diaminohexane spacer group which is attached to biotin's valeric acid side chain, may be modified at the terminal amino group of the spacer via an amide linkage with the acid precursor of SPDP to create a terminal, sulfhydryl-reactive group. The pyridyl disulfide end of biotin-HPDP may react with free thiol groups to form a disulfide bond with loss of pyridine-2-thione.

[0231] Polypeptide modifications set forth herein can be performed in complex samples, including for example, samples having some or all of a proteome. Conditions can be deployed to achieve selective modification of a given type of amino acids. For example, IA-alkyne or EBX2-alkyne can be used to selectively modify cysteines; 2,3 STP-alkyne, ArSq-alkyne or EBA-alkyne can be used to selectively modify lysines; SuTEx2-alkyne or PTAD-alkyne can be used to selectively modify tyrosines; MeTet-alkyne, HC-alkyne or Az-alkyne can be used to selectively modify aspartates and glutamates; CP-alkyne, HMN-alkyne, or MMP-alkyne can be used to selectively modify tryptophans; CP-alkyne can be used to selectively modify histidines, and PhGO-alkyne can be used to selectively modify arginines within a proteome sample, for example, as set forth in Zanon et al., 10.33774 / chemrxiv-2021-w7rss-v2 (2021). A polypeptide may be attached to a particle by a covalent bond or a non-covalent bond. The attachment can occur between a reactive moiety on the polypeptide and a reactive moiety on the particle. The reactive moiety on the polypeptide can be endogenous to the polypeptide, for example, being a reactive moiety of an amino acid side chain group. Alternatively, the reactive moiety can be exogenous to the polypeptide, for example, being produced by functionalization of the polypeptide. Similarly, the reactive moiety on the particle can be endogenous to the structure of the particle or an exogenous moiety that is a modification or addition to the composition of the particle. Reactive moieties on a polypeptide or particle can participate in forming a covalent or non-covalent bond between a particle and polypeptide. Exemplary reactive moieties and attachment configurations include, but are not limited to those set forth above in the context of functionalizing polypeptides or those set forth below. Also, set forth below are methods and compositions for modifying polypeptides or particles to include reactive moieties for use in various attachment configurations.

[0232] Any of a variety of covalent or non-covalent chemistries can be used to attach a polypeptide to a particle. Attachment of a polypeptide to a particle can employ chemical conjugation, bioconjugation, enzymatic conjugation, photo-conjugation, thermal-conjugation, or a combination thereof. (Spicer et. al., Chemical Reviews, 118:7702-7743 (2018), or Hermanson, "Bioconjugate Techniques", Academic Press; 3rd Edition, 2013 ). Chemistries and methods set forth herein in the context of attaching polypeptides to particles can also be used to functionalize polypeptides to incorporate reactive moieties, functionalize particles to incorporate reactive moieties, or attach particles to other substances such as surfaces, solid supports, sites of an array, or other particles. Optionally, chemistries used in one step of a method set forth herein are orthogonal to chemistries used for other steps. For example, chemistry used to attach polypeptides to particles can be orthogonal to chemistries used to functionalize the polypeptides, functionalize the particles and / or attach the particles to a solid support.

[0233] Bioorthogonal chemistries can facilitate selective modification of polypeptides or selective attachment of polypeptides to particles in complex biological milieus, for example, to prevent non-polypeptide molecules from attaching to particles or quenching desired reactions. However, bioorthogonal chemistries need not be deployed in a method set forth herein, for example, when polypeptides are separated from their native milieu or isolated from other biological components. Accordingly, a wide range of other chemistries can be used. In some cases, polypeptides can be modified or attached to particles in non-aqueous solvents, for example, in situations where the polypeptides need not be in a native state.

[0234] A polypeptide can be attached to a particle using a bioorthogonal reaction or click chemistry (see, for example, U.S. Pat. Nos. 6,737,236 and 7,427,678 ); azide alkyne Huisgen cycloaddition reactions, which use a copper catalyst (see, for example, U.S. Pat. Nos. 7,375,234 and 7,763,736 ); Copper-free Huisgen reactions ("metal-free click") using strained alkynes or triazine-hydrazine moieties which can link to aldehyde moieties (see, for example, U.S. Pat. No. 7,259,258 ); triazine chloride moieties which can link to amine moieties; carboxylic acid moieties which can link to amine moieties using a coupling reagent, such as EDC; thiol moieties which can link to thiol moieties; alkene moieties which can link to dialkene moieties that are coupled through Diels-Alder reactions; and acetyl bromide moieties which can link to thiophosphate moieties (see, for example, WO 2005 / 065814 ). A functional group may be configured to react via a click reaction (e.g., metal-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted azide-nitrone cycloaddition, strained alkene reactions, thiol-ene reaction, Diels-Alder reaction, inverse electron demand Diels-Alder reaction, [3+2] cycloaddition, [4+1] cycloaddition, nucleophilic substitution, dihydroxylation, thiol-yne reaction, photoclick, nitrone dipole cycloaddition, norbornene cycloaddition, oxanobornadiene cycloaddition, tetrazine ligation, tetrazole photoclick reactions). Exemplary silane-derivative click reactants may include alkenes, alkynes, azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines (e.g., dibenzocyclooctyne - azide, methyltetrazine - transcyclooctylene, epoxide - thiol, etc.). A click reaction can provide an advantageous method of rapidly forming a bond under biologically conducive conditions (e.g., room temperature, aqueous solvents).

[0235] Copper-Catalyzed Azide-Alkyne Cycloadditions (CuAAC) can be utilized for attachment of two substances, such as attachment of a particle to a polypeptide. In some cases, the (3 + 2) cycloaddition between an azide and alkyne can yield a mixture of two triazole isomers. To achieve conjugation via CuAAC, a copper(I) catalyst can either be added directly, or generated in situ by reduction of an initial copper(II) complex, for example, using ascorbic acid.

[0236] Strain-Promoted Azide-Alkyne Cycloadditions (SPAAC) may be utilized for attachment of two substances, such as attachment of a particle to a polypeptide. Highly strained cyclooctynes can react with azides to form triazoles. In some cases, supramolecular host-guest interactions can also be used to promote azide-alkyne cycloaddition.

[0237] Inverse-electron demand Diels-Alder reactions (IEDDA) may be utilized for attachment of two substances, such as attachment of a particle to a polypeptide. For example, an IEDDA reaction between 1,2,4,5-tetrazines and strained alkenes or alkynes may be employed. Useful reactive moieties include, for example, strained trans-cyclooctenes, functionalized norbornene derivatives, triazines, or spirohexene. In some cases, hetero-Diels-Alder cycloaddition of maleimides and furans may be utilized for attachment. A particularly useful reaction occurs between methyltetrazine (mTz) and transcyclooctene to yield a dihydropyridazine linkage, which may isomerize to a corresponding 1,4-dihydro-isomers or be oxidized to give a pyridazine product. In some cases, oxime and hydrazone may be utilized for attachment of two substances, such as attachment of a particle to a polypeptide. For example, attachment via hydrazone formation can be achieved via difunctional crosslinking.

[0238] In some cases, a Diels-Alder reaction can involve covalent coupling of a diene with an alkene to form a six-membered ring.

[0239] In some cases, transition metal complexes may be utilized for attachment of two substances, such as attachment of a particle to a polypeptide. The nature of late transition metals may make a transition metal complex well suited to the manipulation of unsaturated and polarizable functional groups (olefins, alkynes, aryl iodides, arylboronic acids, etc.). For example, a Pd(0)-catalyst can be used to mediate allyl carbamate deprotections or Suzuki-Miyaura cross-coupling. In other examples, a ruthenium catalyst may be used. For example, with ruthenium complexes, S-allylcysteine can be introduced into polypeptides by a variety of methods, including conjugate addition of allyl thiol to dehydroalanine, direct allylation of cysteine, desulfurization of allyl disulfide, or metabolic incorporation as a methionine surrogate in methionine auxotrophic E. coli.

[0240] In some cases, complex formation with boronic acid derivatives may be used for attachment of substances, such as attachment of a particle to a polypeptide. For example, boronic acid derivatives are able to form ring structures with other molecules having neighboring functional groups consisting of 1,2- or 1,3-diols, 1,2- or 1,3-hydroxy acids, 1,2- or 1,3-hydroxylamines, 1-2- or 1,3-hydroxyamides, 1,2- or 1,3-hydroxyoximes, as well as various sugars or biomolecules containing these species.

[0241] In some cases, enzyme-mediated conjugation may be utilized to attach substances, such as attachment of a particle to a polypeptide. Enzyme-mediated conjugation may proceed via transglutaminases, peroxidases, sortase, SpyTag-SpyCatcher, or a combination thereof. Photo conjugation and activation may proceed via photoacrylate crosslinking reaction, photo thiol-ene reaction, photo thiol-yne reaction, or a combination thereof. In some cases, attachment or conjugation may proceed via noncovalent interactions, these may be through self-assembling peptides, binding sequences, host-guest chemistry, complementary hybridization of nucleic acids, or a combination thereof.

[0242] A polypeptide can be attached to a particle by a receptor-ligand binding interaction. For example, binding of (strept)avidin to the small molecule biotin may be used. (Strept)avidin may be attached to a first substance, such as a particle, and biotin may be attached to a second substance, such as a polypeptide, thereby allowing the substances to become attached via binding of the (strept)avidin to the biotin. Other receptor ligand pairs that can be used instead of (strept)avidin-biotin include, but are not limited to, antibodies and their epitopes, aptamers and their epitopes, complementary nucleic acid molecules, lectins and carbohydrates, or nucleic acids and nucleic acid binding polypeptides. Further examples of useful receptor-ligand pairs include probes set forth herein and the targets to which they bind. A polypeptide, particle or other substance set forth herein can be functionalized to include a receptor moiety or ligand moiety, for example, using functionalization chemistries set forth herein.

[0243] A reactive handle comprising a functional group may be coupled or conjugated to a polypeptide by a molecule comprising a linking group. A linking group may include molecules or macromolecules that increase the length or size of the functionalization groups. A linking group may include homobifunctional linkers, hetereobifunctional linkers, or other polyfunctional linkers. A linking group may be coupled or conjugated to a polypeptide before a second molecule is coupled or conjugated to the linking groups. A linking group may comprise a flexible region (e.g., a PEG linker) or may be a rigid linker (e.g., a polyunsaturated alkyl group). A rigid linker may facilitate controlling the orientation of a polypeptide when coupled to an anchoring group. A linking group may incorporate a detectable label that facilitates measurement of functional group incorporation or extent of functionalization. For example, an incorporated group in a linking group may comprise a molecule with a measurable and / or quantifiable absorbance (e.g., chromophores) or emission signal (e.g., fluorophores, radiolabels, isotopes, etc.)

[0244] A linking group may incorporate a reporting molecule. A reporting molecule may comprise a molecule that is formed or released upon functionalization of a polypeptide. For example, a polypeptide may be functionalized with a functionalizing compound comprising a fluorescent leaving group (e.g., NHS ester coupled to a fluorophore). In some cases, released reporting molecules may be measured to quantify an extent of functionalization.

[0245] A functionalized polypeptide fraction may be prepared and / or stored in a functionalized polypeptide solvent composition. A functionalized polypeptide solvent composition may comprise one or more polypeptide composites in solution or suspension. A functionalized polypeptide solvent composition may be formulated to be a homogeneous liquid medium. A functionalized polypeptide solvent composition may be formulated to be a single-phase liquid medium. A functionalized polypeptide solvent composition may be formulated to be a multi-phase liquid medium, such as an oil-in-water emulsion or a water-in-oil emulsion. For a functionalized polypeptide solvent composition formulated as an emulsion, anchoring groups or polypeptide composites may be solvated or suspended within the dissolved phase. In some cases, a functionalized polypeptide composition may be stored in a non-fluid phase, such as polypeptide immobilized on a solid, encapsulated polypeptides (e.g., alginates), or dried or lyophilized polypeptides.

[0246] A functionalized polypeptide fraction may comprise a functionalization internal standard. The functionalization internal standard may comprise a plurality of functionalization standard polypeptides. Functionalization standard polypeptides may include polypeptides that undergo functionalization in the same reaction as sample polypeptides. Functionalization standard polypeptides may include polypeptides that were functionalized in a different setting than the sample polypeptides by the same reaction as the sample polypeptides. Functionalization standard polypeptides may include functionalized polypeptides that have had functional groups added by a different method than the method utilized to functionalize the sample polypeptides. A functionalization internal standard may also include non-polypeptide components that may be used to determine the conditions of a polypeptide functionalization method, such as pH indicator dyes or competitor reagents.Anchoring Groups and Polypeptide Coupling

[0247] Polypeptides may be coupled or conjugated to other molecules or materials. The polypeptide(s) can be sample polypeptides, standard polypeptides or other polypeptides, such as those set forth herein. The sample polypeptides can be from a source, sample or fraction including, for example, those set forth herein. In some cases, a polypeptide may be coupled or conjugated to other molecules or materials during or after a polypeptide functionalization reaction. In other cases, a polypeptide may be coupled or conjugated to other molecules or materials without a polypeptide functionalization reaction. For example, a polypeptide within a cell may be directly coupled to a molecule or material. A polypeptide may be coupled or conjugated to another molecule or material before or after the polypeptide has been separated from a sample comprising the polypeptide. The methods and compositions set forth below may generally be exemplified with reference to a functionalized polypeptide; however, it will be understood that the examples can be extended to a population having the same species of functionalized polypeptide, a population having the same species of unfunctionalized polypeptide, a population having different species of functionalized polypeptide, or a population having different species of unfunctionalized polypeptide.

[0248] Multiple functionalized or unfunctionalized polypeptides, whether being the same or different species, can be attached to the same molecule or material, for example, the functionalized polypeptides can be attached to a surface to form an array of addresses where each address has a different coupled or conjugated molecule attached or the functionalized polypeptides can be attached to a molecule such as a polymer having multiple attachment sites. Alternatively, multiple functionalized or unfunctionalized polypeptides, whether being the same or different species, can be attached to a population of molecules, beads or particles such that each molecule, bead or particle is attached to a different functionalized or unfunctionalized polypeptide. Moreover, methods and compositions may be exemplified below with reference to proteins; however, it will be understood that the examples can be extended to other polypeptides.

[0249] The addition of one or more functional groups on a polypeptide molecule may facilitate the coupling or conjugation of the polypeptide molecule to another molecule or material. In some cases, a polypeptide may be coupled or conjugated to one or more additional molecules to form a polypeptide composite. A polypeptide composite may be formed with a small molecule such as a fluorophore, or a large molecule such as another biomolecule (e.g., polypeptide or nucleic acid). The polypeptide composite may alter the solution properties of a polypeptide from which it is formed. A polypeptide composite may increase or decrease the solubility of a polypeptide, or may render a polypeptide amphiphilic by drawing it toward a liquid / gas or liquid / liquid interface. A polypeptide composite may be formed for any necessary purpose, including localizing or depositing a polypeptide at a surface or adding a detection label to a polypeptide (e.g., fluorophore, radiolabel, nucleic acid tag, streptavidin tag). In some cases, a polypeptide composite may permit characterization of a polypeptide by adding a new functionality or creating a new possible interaction with the polypeptide. For example, a horseradish peroxidase enzyme may be coupled or conjugated to each added functional group on a polypeptide, thereby permitting polypeptide size to be estimated by the activity level of the enzyme.

[0250] In some cases, a polypeptide or a plurality of polypeptides may be directly coupled or conjugated onto a solid substrate surface such as a surface at an address of an array, or a surface of a flow cell, microwell or microbead. For example, a polypeptide that has been functionalized with an NHS group may be contacted with a silicon surface covered in an aminated silane monolayer, thereby forming a covalent bond between the polypeptide and the silane. In other cases, a polypeptide may be functionalized with a magnetic nanoparticle that is configured to couple or conjugate with a complementary magnetic material (e.g., a surface with an array of embedded or tethered magnetic nanoparticles).

[0251] Polypeptides from a functionalized or unfunctionalized polypeptide fraction may be coupled or conjugated to one or more anchoring groups. An anchoring group may comprise a particle that mediates or facilitates the binding of the polypeptide to a substrate or surface. An anchoring group may comprise a particle that couples a sample polypeptide to a solid support. An anchoring group may comprise a particle such as a nucleic acid particle, a polypeptide, a polymer, an inorganic nanoparticle, an organic nanoparticle, or a combination thereof. An anchoring group may interact with a surface by an interaction such as electrostatic adhesion, magnetic adhesion, covalent bonding, ionic bonding, hydrogen bonding, or coordinate bonding. An anchoring group may interact with a surface in a reversible fashion or an irreversible fashion.

[0252] A polypeptide (e.g. a sample polypeptide or standard polypeptide) from a plurality of sample polypeptides may be coupled or conjugated to an anchoring group by a reversible or irreversible interaction. A polypeptide of a plurality of polypeptides may be coupled to an anchoring group of a plurality of anchoring groups by a covalent bond. In some configurations, a polypeptide of a plurality of polypeptides may be coupled to an anchoring group of a plurality of anchoring groups by a click reaction or other covalent coupling chemistry exemplified elsewhere herein. A polypeptide of a plurality of polypeptides may be coupled to an anchoring group of a plurality of anchoring groups by a non-covalent interaction. In some configurations, the non-covalent interaction may be an electrostatic interaction, magnetic interaction, a hydrogen bond, or a binding interaction. In some configurations, the non-covalent hydrogen bond interaction may comprise nucleic acid hybridization. In other configurations, the non-covalent binding interaction may comprise a receptor-ligand interaction or a receptor-small molecule interaction, such as streptavidin-biotin, FITC-anti-FITC antibody, or digoxigenin-anti-digoxigenin antibody or other non-covalent interaction exemplified elsewhere herein.

[0253] An anchoring group may comprise a macromolecule or particle that possesses a positive or negative overall surface charge density. An anchoring group may comprise a macromolecule or particle that possesses a positive or negative region of surface charge density. The surface charge density of an anchoring group may be the opposite charge of a surface that a polypeptide conjugate is to be deposited upon. The surface charge density of an anchoring group may be neutral. The surface charge density of an anchoring group may be uniform over the available surface area of the anchoring group. A uniform surface charge density may increase the speed and / or likelihood of the anchoring group depositing upon a surface or material. Regions of positive or negative surface charge density of an anchoring group may be localized to one or more regions of the anchoring group structure. Localized surface charge density on an anchoring group may cause a polypeptide conjugate containing the anchoring group to deposit on a surface or material with a uniform or controlled orientation. Surface charge density of an anchoring group or a polypeptide conjugate containing an anchoring group may be measured by a suitable method such as electrophoretic measurement of zeta potential. A surface charge density of an anchoring group or a polypeptide conjugate containing an anchoring group may be determined by experimental measurement, computational modeling, or a combination thereof.

[0254] Anchoring groups may comprise one or more macromolecules. A suitable macromolecule for an anchoring group may include a macromolecule with a uniform or localized region of positive or negative surface charge density. A macromolecule in an anchoring group may possess a controlled or engineered structure, including a feature such as a polypeptide coupling or conjugation site, or a surface bonding site. A polypeptide coupling or conjugation site on an anchoring group may comprise a functional group configured to react with a functional group of a functionalized or unfunctionalized polypeptide, thereby forming a covalent bond between the anchoring group and the polypeptide. Suitable macromolecules may include nucleic acids, proteins, or polymers. A nucleic acid anchoring group may comprise a structured nucleic acid particle (SNAP) such as a DNA nanoball, DNA nanotube, or DNA origami. A polypeptide-based anchoring group may include an engineered or non-engineered polypeptide that has a tendency to deposit on a surface or material. A polypeptide for a polypeptide-based anchoring group may be prepared for conjugation to a polypeptide from a polypeptide fraction by methods similar to those described above. A polymer-based anchoring group may include ionic or nonionic polymers.

[0255] In other configurations, an anchoring group may comprise a particle, such as a nanoparticle, that provides a plurality of attachment sites for two or more binding components, and optionally one or more label components. In some configurations, a particle may comprise a surface that is functionalized, can be functionalized, or is otherwise modifiable to provide attachment sites for polypeptide coupling. In some configurations, a particle may provide a template for a shell, surface coating, or surface layer (e.g., a surface coating comprising a polymer or hydrogel coating, a surface layer of functional groups) that contains or can be modified to contain attachment sites for detectable probe components. A surface coating may comprise a polymer, biopolymer, metal, or metal oxide. In some configurations, an anchoring group may effectively function as a label component (e.g., a fluosphere or quantum dot). A particle for an anchoring group may comprise a surface coating or surface layer that comprises a surface electrical charge. The surface electrical charge may comprise a net positive charge or a net negative charge. An anchoring group may be formulated or modified to comprise a plurality of functional groups that are configured to couple to a solid support by a covalent or non-covalent interaction. In some configurations, the plurality of functional groups may comprise a functional group selected from the group consisting of an alkyl, alkenyl, alkynyl, phenyl, halide, hydroxyl, carbonyl, aldehyde, acyl halide, ester, carboxylate, carboxyl, carboalkoxy, methoxy, hydroperoxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, epoxide, carboxylic anhydride, carboxamide, amine, ketimine, aldimine, imide, azide, azo, cyanate, isocyanate, nitrate, nitrile, isonitrile, nitrosoxy, nitro, nitroso, oxime, pyridyl, carbamate, sulfhydryl, sulfide, disulfide, sulfinyl, sulfonyl, sulfinom, sulfo, thiocyanate, isothiocyanate, carbonothioyl, thioester, thionoester, phosphino, phosphono, phosphonate, phosphate, borono, boronate, and a borinate. In some configurations, an anchoring group may be modified to comprise a functional group that is configured to undergo a click reaction. In other configurations, an anchoring group may be modified to comprise a functional group that is configured to undergo a chemical cross-linking or a photo-initiated cross-linking reaction.

[0256] An anchoring group may comprise a detectable label that permits detection of the anchoring group. A detectable label may comprise a fluorescent label, a luminescent label, a radiolabel, an enzymatic tag, or a nucleic acid label or barcode. An anchoring group may be conjugated with a detectable label. The conjugated detectable label may be conjugated by a covalent bond (e.g., a reactive dye) or a non-covalent interaction (e.g., hybridization of a nucleic acid tag, an intercalation dye). A detectable label may be used to quantify anchoring groups in solution or detect anchoring groups at individual locations on a substrate.

[0257] An anchoring group or a linkage between an anchoring group and a polypeptide may further comprise a linker. A linker may comprise a bifunctional, trifunctional, or polyfunctional linker. A bifunctional linker may comprise a homobifunctional linker or a heterobifunctional linker. A linker may include a reporting molecule that is released upon successful coupling of an anchoring group to a polypeptide. For example, a click-to-release strategy may be utilized to covalently couple a polypeptide comprising a click handle with an anchoring group comprising a second click handle (e.g., inverse-electron demand Diels Alder click-to-release).

[0258] An anchoring group may be configured to be coupled or conjugated to a functionalized or unfunctionalized polypeptide (e.g. sample polypeptide or standard polypeptide). Functional groups capable of rapidly forming covalent bonds with functionalized or unfunctionalized polypeptides may be of particular interest for the functionalization of anchoring groups. In general, functional groups of interest will include most common species for bioconjugation. Such functional groups may include "click" reagents that are capable of forming highly specific products with complementary functional groups in a rapid and irreversible fashion. Exemplary functionalization chemistries are described above. An anchoring group may comprise one or more sites for polypeptide coupling or conjugation. An anchoring group with more than one attachment site may be capable of coupling or conjugating more than one polypeptide. An anchoring group may comprise a fixed number of polypeptide attachment sites, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, or more than 1000000 attachment sites. An anchoring group may comprise a fixed number of polypeptide attachment sites, such as at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, or more than 1000000 attachment sites. Alternatively or additionally, an anchoring group may comprise a fixed number of polypeptide attachment sites, such as no more than about 1000000, 500000, 100000, 50000, 10000, 5000, 1000, 500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 attachment sites.

[0259] A coupling or conjugation reaction between a functionalized polypeptide and an anchoring group may be performed in a fashion that yields substantially complete coupling or conjugation of all polypeptides, all anchoring groups, or a combination of both. For example, polypeptide may be supplied in excess such that nearly all anchoring groups become coupled or conjugated to a polypeptide by completion of the reaction. Alternatively, an anchoring group may be supplied in excess such that all polypeptides in a polypeptide fraction become coupled or conjugated to an anchoring group by completion of the reaction. A coupling or conjugation reaction may be performed with a polypeptide: anchoring group ratio of about 1:1000, 1:500, 1:250, 1:100, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 25:1, 30:1, 50:1, 100:1, 250:1, 500:1, 1000:1, or more. A conjugation reaction may be performed with a polypeptide: anchoring group ratio of at least about 1:1000, 1:500, 1:250, 1:100, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 25:1, 30:1, 50:1, 100:1, 250:1, 500:1, 1000:1, or more. Alternatively or additionally, a conjugation reaction may be performed with a polypeptide:anchoring group ratio of no more than about 1000:1, 500:1, 250:1, 100:1, 50:1, 30:1, 25:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500, 1:1000, or more.

[0260] A conjugation reaction between a functionalized or unfunctionalized polypeptide (e.g., sample polypeptide or standard polypeptide) and an anchoring group may occur at a fixed temperature. In some cases, a coupling or conjugation reaction may occur at a first temperature for a fixed amount of time, then a second temperature for a fixed amount of time. A coupling or conjugation reaction between a functionalized or unfunctionalized polypeptide and an anchoring group may occur at a temperature of about - 80 °C, - 70 °C, - 60 °C, - 50 °C, - 40 °C, - 30 °C, - 20 °C, - 10 °C, - 5 °C, 0 °C, 4 °C, 10 °C, 20 °C, 30 °C, 37 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, or about 95 °C. A coupling or conjugation reaction between a functionalized polypeptide and an anchoring group may occur at a temperature of at least about - 80 °C, - 70 °C, - 60 °C, - 50 °C, - 40 °C, -30 °C, -20 °C, -10 °C, -5 °C, 0 °C, 4 °C, 10 °C, 20°C, 30°C, 37 °C, 40 °C, 50 °C, 60°C, 70 °C, 80 °C, 90 °C, or about 95 °C. Alternatively or additionally, a conjugation reaction between a functionalized or unfunctionalized polypeptide and an anchoring group may occur at a temperature of no more than about 95 °C, 90 °C, 80 °C, 70 °C, 60 °C, 50 °C, 40 °C, 37 °C, 30 °C, 20 °C, 10 °C, 4 °C, 0 °C, - 5 °C, - 10 °C, - 20 °C, - 30 °C, - 40 °C, - 50 °C, -60 °C, - 70 °C, or about - 80 °C.

[0261] A conjugation reaction between a functionalized or unfunctionalized polypeptide (e.g., sample polypeptide or standard polypeptide) and an anchoring group may occur for a fixed amount of time. A conjugation reaction between a functionalized or unfunctionalized polypeptide and an anchoring group may occur for about 1 min, 30 mins, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 wks, 3 wks, 4 wks, 1 mth, or more than 1 mth. A conjugation reaction between a functionalized or unfunctionalized polypeptide and an anchoring group may occur for at least about 1 min, 30 mins, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 wks, 3 wks, 4 wks, 1 mth, or more than 1 mth. Alternatively or additionally, a conjugation reaction between a functionalized or unfunctionalized polypeptide and an anchoring group may occur for no more than about 1 month, 4 wks, 3 wks, 2 wks, 10 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 23 hrs, 22 hrs, 21 hrs, 20 hrs, 19 hrs, 18 hrs, 17 hrs, 16 hrs, 15 hrs, 14 hrs, 13 hrs, 12 hrs, 11 hrs, 10 hrs, 9 hrs, 8 hrs, 7 hrs, 6 hrs, 5 hrs, 4 hrs, 3 hrs, 2 hrs, 1 hr, 30 mins, 1 min, or less than 1 min.

[0262] An anchoring group may be coupled or conjugated to a functionalized or unfunctionalized polypeptide (e.g., sample polypeptide or standard polypeptide). An anchoring group may be coupled or conjugated to a functionalized polypeptide before or after the anchoring group has been deposited on a surface. FIG. 9A depicts a broad scheme for forming a polypeptide composite then depositing the polypeptide composite on a surface or material. A polypeptide 910 comprising a first functional group 920 may be contacted with an anchoring group 940 containing a second functional group 930. The first functional group 920 may be configured to form a covalent bond with the second functional group 930. The reaction between the first functional group 920 and the second functional group 930 forms a polypeptide composite 950. The reaction between the polypeptide 910 and the anchoring group 940 may occur in the presence of a detergent. The reaction between the polypeptide 910 and the anchoring group 940 may occur in the presence of a standard polypeptide, such as a coupling standard polypeptide. After a polypeptide composite 950 has been formed, the polypeptide composite 950 may be contacted with a surface or material 960 to deposit the polypeptide composite 950 on the surface or material 960. The polypeptide composite 950 may become attached to the surface or material 960 by a covalent or non-covalent interaction. The deposition of the polypeptide composite 950 on the surface or material 960 may occur in the presence of a detergent. The deposition of the polypeptide composite 950 on the surface or material 960 may occur in the presence of a standard polypeptide, such as a deposition standard polypeptide.

[0263] FIG. 9B depicts an alternate scheme for forming a polypeptide composite. An anchoring group 940 comprising a second functional group 930 is brought in contact with a surface or material 960. The anchoring group 940 may become coupled to the surface or material 960 by a covalent or non-covalent interaction. The deposition of the anchoring group 950 on the surface or material 960 may occur in the presence of a detergent. The deposition of the anchoring group 950 on the surface or material 960 may occur in the presence of a standard polypeptide, such as a deposition standard polypeptide. The deposited anchoring group 940 on the surface or material 960 may be subsequently contacted with a polypeptide 910 containing a first functional group 920. The first functional group 920 may be configured to form a covalent bond with the second functional group 930. The reaction between the first functional group 920 and the second functional group 930 forms a polypeptide composite 950 that is deposited on a surface or material 960. The reaction between the polypeptide 910 and the anchoring group 940 may occur in the presence of a detergent. The reaction between the polypeptide 910 and the anchoring group 940 may occur in the presence of a standard polypeptide, such as a coupling standard polypeptide.

[0264] A polypeptide composite (e.g., sample polypeptide composite or standard polypeptide composite) may be prepared and / or stored in a polypeptide composite solvent composition. A polypeptide composite solvent composition may comprise one or more polypeptide composites in solution or suspension. A polypeptide composite solvent composition may be formulated to be a homogeneous liquid medium. A polypeptide composite solvent composition may be formulated to be a single-phase liquid medium. A polypeptide composite solvent composition may be formulated to be a multi-phase liquid medium, such as an oil-in-water emulsion or a water-in-oil emulsion. For a polypeptide composite solvent composition formulated as an emulsion, anchoring groups or polypeptide composites may be solvated or suspended within the dissolved phase.

[0265] A polypeptide composite solvent composition may comprise a stable solution or suspension of polypeptide composites (e.g., sample polypeptide composites or standard polypeptide composites). A stable solution or suspension of polypeptide composites may exhibit no detectable sedimentation or agglomeration of polypeptide composites over a fixed time period such as about 1 min, 5 mins, 10 mins, 30 mins, 1 hr, 2 hrs, 3 hrs, 6 hrs, 12 hrs, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 wks, 3 wks, 4 wks, 1 mth, 3 mths, 6 mths, 1 yr, or more than 1 yr.

[0266] A polypeptide conjugate solvent composition may be utilized for the storage of prepared polypeptide conjugates(e.g., sample polypeptide composites or standard polypeptide composites). A polypeptide conjugate solvent composition may be utilized for the deposition of polypeptide conjugates on a substrate. In some cases, polypeptide conjugates may be deposited on a substrate in a polypeptide conjugate solvent composition without further modification of the solvent composition. In other cases, polypeptide conjugates may be deposited on a substrate in a polypeptide conjugate solvent composition that is modified at the time of deposition (e.g., adding a component, adjusting a component concentration, changing the composition pH). In other cases, a polypeptide conjugate solvent composition may be exchanged (e.g., by dialysis, filtration, or extraction) for a deposition solvent composition before polypeptide conjugates are deposited on a substrate.

[0267] A coupled or conjugated polypeptide fraction may comprise a coupling internal standard. The coupling internal standard may comprise a one or a plurality of coupling standard polypeptides. Coupling standard polypeptides may include polypeptides that undergo coupling by the same coupling method as sample polypeptides. Coupling standard polypeptides may include polype...

Claims

1. A method of forming a polypeptide array, comprising: (a) providing a sample comprising sample polypeptides; (b) separating the sample polypeptides from the sample in the presence of separation standard polypeptides, wherein the separation standard polypeptides comprise polypeptides derived from a differing source than the sample polypeptides, and wherein each separation standard polypeptide of the separation standard polypeptides comprises a universal and / or unique tag, wherein the universal tag distinguishes the separation standard polypeptides from other polypeptides in the method and wherein the unique tag distinguishes a separation standard polypeptide from all other separation standard polypeptides in the method, wherein the tag is a peptide sequence; (c) coupling the sample polypeptides to anchoring groups to form sample polypeptide composites, wherein the coupling occurs in the presence of coupling standard polypeptides, wherein the coupling standard polypeptides comprise polypeptides derived from a differing source than the sample polypeptides, and wherein each coupling standard polypeptide of the coupling standard polypeptides comprises a universal and / or unique tag, wherein the universal tag distinguishes the coupling standard polypeptides from other polypeptides in the method and wherein the unique tag distinguishes a coupling standard polypeptide from all other coupling standard polypeptides in the method, wherein the tag is a peptide sequence; and (d) attaching at least a fraction of the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides to a solid support, whereby each of the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides that is attached to the solid support comprises an address of the polypeptide array.

2. The method of claim 1, wherein the attaching of step (d) occurs in the presence of attachment standard polypeptides, whereby each of the attachment standards that is attached to the solid support comprises an address of the polypeptide array.

3. The method of claim 1 or claim 2, wherein the attachment standard polypeptides comprise attachment standard polypeptide composites, wherein each of the attachment standard polypeptide composites comprises an attachment standard polypeptide coupled to an anchoring group.

4. The method of any one of the preceding claims, wherein the separation standard polypeptides comprise separation standard polypeptide composites, wherein each of the separation standard polypeptide composites comprises a separation standard polypeptide coupled to an anchoring group.

5. The method of any one of the preceding claims, wherein the coupling standard polypeptides comprise coupling standard polypeptide composites, wherein each of the coupling standard polypeptide composites comprises a coupling standard polypeptide coupled to an anchoring group.

6. The method of any one of the preceding claims, wherein the separation standard polypeptides, coupling standard polypeptides or attachment standard polypeptides comprise a synthetic polypeptide, a non-natural amino acid or a synthetically modified amino acid.

7. The method of any one of the preceding claims, wherein the polypeptide array comprises at least 1 x 104 different full-length primary protein structures.

8. The method of any one of the preceding claims, wherein the anchoring groups comprise particles configured to couple to the solid support, optionally wherein the particles comprise a nucleic acid, a polypeptide, a polymer, an inorganic nanoparticle, an organic nanoparticle, a structured nucleic acid particle (SNAP), a nucleic acid origami or a nucleic acid nanoball.

9. The method of any one of the preceding claims, further comprising: (e) detecting the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides at addresses of the polypeptide array.

10. The method of claim 9, further comprising detecting the attachment standard polypeptides at addresses of the polypeptide array.

11. The method of claim 9, wherein the sample polypeptide composites, the separation standard polypeptides and the coupling standard polypeptides are detected as single polypeptide resolution.

12. The method of any one of claims 9-11, further comprising quantifying the amount of a particular sample polypeptide species in the sample by comparing (i) a number of the addresses attached to a separation standard polypeptide with (ii) the number of the addresses attached to a sample polypeptide composite that comprise the particular sample polypeptide species.

13. The method of any one of claims 9-12, further comprising quantifying the amount of a particular sample polypeptide species in the sample by comparing (i) a number of addresses attached to a coupling standard polypeptide with (ii) a number of addresses attached to a sample polypeptide composite that comprise the particular sample polypeptide species.

14. The method of any one of claims 9-13, further comprising quantifying the amount of a particular sample polypeptide species in the sample by comparing (i) a number of addresses attached to an attachment standard polypeptide with (ii) a number of addresses attached to a sample polypeptide composite that comprise the particular sample polypeptide species.