Peptide purification formulations and methods

Abstract

Improved formulations for the purification of peptides from biological samples, and methods and kits for purifying peptides from biological samples (e.g., cells and tissues), and the use of purified peptides (e.g., polypeptides derived from protein digests) in mass spectrometry (e.g., LC-MS) applications are described.

Description

[Technical Field]

[0001] Formulations for the purification of peptides from biological samples, and methods and kits for purifying peptides (e.g., polypeptides derived from protein digests) from biological samples, and the use of purified peptides in downstream applications such as mass spectrometry, are described. [Background technology]

[0002] Mass spectrometry (MS) is a powerful tool for analyzing proteins and their amino acid sequences, including post-translational modifications. A typical method for preparing protein samples for MS analysis is to first digest the protein with enzymes (e.g., trypsin, Lys-C, etc.) to generate a mixture of smaller peptide fragments, and then analyze the fragments on an LC-MS system to extract qualitative and / or quantitative proteomic information (referred to herein as a "bottom-up" proteomics workflow). A typical "bottom-up" proteomics workflow generally requires proteins to be extracted from biological samples prior to digestion. Biological samples (e.g., tissues, cultured cells, and biological fluids) are complex matrices containing a variety of small molecules, carbohydrates, lipids, nucleic acids, salts, peptides, and proteins. Many of these components are innate to the biological sample, while some are added during sample preparation to improve protein solubility, introduce desired chemical modifications into proteins / peptides, or maintain an optimal pH for proteolytic activity during protein digestion. To prevent chromatographic interference, ionization suppression, and clogging of LC plumbing components, it is important to remove as many non-peptide species as possible from the sample matrix before LC-MS analysis. This cleanup step is commonly known as "sample desalting." Traditionally, this procedure is performed on a reversed-phase resin using solid-phase extraction (SPE) chromatography, in which the sample is first acidified and then loaded onto the resin in aqueous solution. Once the sample is loaded onto the resin, the resin is washed with aqueous solution to remove small hydrophilic molecules and ions. Peptides remain bound to the resin through hydrophobic interactions and are eluted using an acidic solution containing an organic solvent (e.g., 50% acetonitrile in water). A drawback of this desalting method is that hydrophobic molecules, such as lipids and surfactants, also bind to the resin and co-elute with the peptide sample. This results in contamination of the peptide sample with hydrophobic molecules, which can lead to poor data quality and damage to the LC equipment.Thus, there is a need for improved formulations, alternative stationary phases, and desalting solution formulations and methods for purifying proteins and peptides from biological samples so that the proteins and peptides are sufficiently pure for high quality analysis by LC-MS. Summary of the Invention

[0003] In one aspect, methods and kits are disclosed herein for use in purifying samples containing peptides. Samples include those of biological origin, synthetically prepared samples, and combinations thereof. In some embodiments, the peptides contain 2 to 50 amino acid residues. In some embodiments, the sample contains two or more peptides. In some embodiments, the sample is a digested protein or polypeptide. The sample may further contain salts, detergents, lipids, small neutral molecules, or combinations thereof. The sample may further contain one or more contaminants. The contaminants may be hydrophilic contaminants, such as neutral contaminants, anionic contaminants, or combinations thereof. In some embodiments, the sample contains hydrophobic contaminants, such as lipids or detergents. In some embodiments, the hydrophilic contaminants are salts, nucleic acids, or carbohydrates. In some embodiments, the sample comprises a monovalent cationic contaminant such as ammonium, sodium, potassium, tris(hydroxymethyl)aminomethane (tris), hydroxylamine, ethanolamine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), glycine, a mass tag, or a combination thereof. In some embodiments, the monovalent cationic contaminant is or comprises a mass tag or a derivative thereof.

[0004] In another aspect, there is provided a method of purifying a sample (e.g., a biological sample), comprising: (a) contacting a biological sample with a hydrophobic polymer-based cation exchange material under acidic conditions, such that the sample binds to the cation exchange material, and the sample contains (i) peptides, and (ii) monovalent cationic contaminants and / or hydrophobic contaminants; (b) washing the cation exchange material with an acidic solution, the acidic solution comprising greater than 20% (v / v) of a volatile organic solvent, water, and a volatile salt, such that the peptides are retained on the cation exchange material and monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; (c) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile compound selected from a volatile organic solvent, water, and a volatile base, a volatile salt, and combinations thereof.

[0005] In yet another aspect, there is provided a method of purifying a sample (e.g., a biological sample), comprising: (a) contacting a biological sample with a hydrophobic polymer-based cation exchange material under acidic conditions, such that the sample binds to the cation exchange material, the sample comprising (i) peptides, (ii) monovalent cationic contaminants and / or hydrophobic contaminants, and (iii) hydrophilic contaminants; (b) washing the cation exchange material with a first acidic solution, the first acidic solution comprising greater than 20% (v / v) of a volatile organic solvent, water, and a volatile salt, such that the peptides are retained on the cation exchange material and monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; (c) washing the cation exchange material with a second acidic solution, the second acidic solution comprising 20% ​​(v / v) or less of a volatile organic solvent, water, and a volatile acid, wherein the peptides are retained on the cation exchange material and hydrophilic contaminants are removed;

[0006] (d) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile compound selected from a volatile organic solvent, water, and a volatile base, a volatile salt, and combinations thereof.

[0007] In yet another aspect, there is provided a method of purifying a sample (e.g., a biological sample), comprising: (a) contacting a biological sample with a hydrophobic polymer-based cation exchange material under acidic conditions, such that the sample binds to the cation exchange material, the biological sample containing (i) peptides, (ii) monovalent cationic contaminants and / or hydrophobic contaminants, and (iii) hydrophilic contaminants; (b) washing the cation exchange material with a first acidic solution, the first acidic solution comprising 20% ​​(v / v) or less of a volatile organic solvent, water, and a volatile acid, such that the peptides are retained on the cation exchange material and hydrophilic contaminants are removed from the cation exchange material; (c) washing the cation exchange material with a second acidic solution, the second acidic solution comprising greater than 20% (v / v) of a volatile organic solvent, water, and a volatile salt, wherein the peptides are retained on the cation exchange material and monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; (d) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile compound selected from a volatile organic solvent, water, and a volatile base, a volatile salt, and combinations thereof.

[0008] In any of the methods disclosed herein, the sample can be contacted with a hydrophobic polymer-based cation exchange material at pH 1-5, the cation exchange material can be washed at pH 1-5, and / or the sample can be eluted from the cation exchange material at a pH above 5 (e.g., 5-11). In certain methods, the retained peptides can be eluted from the cation exchange material using an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile organic solvent, water, and a volatile salt. In certain embodiments, the methods disclosed herein can further include treating the biological sample with a proteolytic enzyme (e.g., trypsin or Lys-C) to generate peptides prior to contacting the biological sample with the cation exchange material.

[0009] In yet another aspect, a kit for purifying a biological sample containing peptides is provided. The kit may include (a) a hydrophobic polymer-based cation exchange material, (b) an acidic solution containing a volatile salt, water, and more than 20% (v / v) of a volatile organic solvent, (c) a neutral or alkaline solution containing a volatile base, water, and a volatile organic solvent, and (d) instructions for using the kit to purify the biological sample.

[0010] In yet another aspect, a kit for purifying a biological sample containing peptides is provided. The kit may include: (a) a hydrophobic polymer-based cation exchange material; (b) an acidic solution containing more than 20% (v / v) of a volatile organic solvent, a volatile salt, and water; (c) an acidic solution containing 20% ​​or less (v / v) of a volatile organic solvent, a volatile salt, and water; (d) a neutral or alkaline solution containing a volatile base, water, and a volatile organic solvent; and (e) instructions for using the kit to purify the biological sample.

[0011] In any of the methods and kits provided herein, the hydrophobic polymer-based cation exchange material can be sulfonated divinylbenzene polystyrene, sulfonated polydivinylbenzene, or sulfonated divinylbenzene / polystyrene / pyrrolidone resin. In some embodiments, the volatile salt can include a volatile acid and a volatile base. For example, the volatile acid can be formic acid, acetic acid, trifluoroacetic acid, or trichloroacetic acid. Representative examples of volatile bases include trimethylamine, ammonia, triethylamine, piperidine, and butylamine. In any of the methods and kits provided herein, the volatile organic solvent can be acetonitrile, methanol, ethanol, n-propanol, isopropanol, acetone, or a combination thereof. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram of a peptide purification method using multiple wash solutions. [Figure 2] Figure 2A shows the MS (total ion) chromatograms of HeLa cell protein digests labeled with TMT reagent and cleaned up using Wash B solution containing 0.1% formic acid (Figure 2A), Wash B solution containing 0.1% (v / v) formic acid and 0.1% (v / v) triethylamine (Figure 2B), and Wash B solution containing 0.5% (v / v) formic acid and 0.5% (v / v) triethylamine (Figure 2C). The boxed areas in the chromatograms indicate signals due to quenched and hydrolyzed TMT-derived ions. [Figure 3A] A series of images from a quantitative colorimetric peptide assay for visual and spectroscopic assessment of peptide yield after cleanup of a HeLa cell protein digest labeled with TMT reagent and cleaned up using Wash B solution containing 0.1% formic acid (A), Wash B solution containing 0.1% (v / v) formic acid and 0.1% (v / v) triethylamine (B), and Wash B solution containing 0.5% (v / v) formic acid and 0.5% (v / v) triethylamine (C). [Figure 3B]1 is a bar graph comparing peptide yields for samples processed using the wash solutions described above. Due to the presence of residual TMT reagent, samples cleaned up with Wash B solution (A) produced a signal that was more than five times higher than the signal from samples processed with a buffer containing TEA. [Figure 4A] 1 shows a number of MS / MS spectra acquired from LC-MS analysis of TMT-labeled peptide samples washed with various washing solutions. [Figure 4B] The number of peptide spectral matches (PSMs) identified from LC-MS analysis of TMT-labeled peptide samples washed with various washing solutions is shown. [Figure 4C] The number of unique peptides identified from LC-MS analysis of TMT-labeled peptide samples washed with various washing solutions is shown. [Figure 4D] The number of protein groups identified from LC-MS analysis of TMT-labeled peptide samples washed with various washing solutions is shown. [Figure 5A] 1 shows protein groups identified from LC-MS analysis of various peptide sample amounts washed with various washing solutions. [Figure 5B] Unique peptides identified from LC-MS analysis of various peptide sample amounts washed with various wash solutions are shown. DETAILED DESCRIPTION OF THE INVENTION

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All patents, applications, published applications, and other publications mentioned herein are incorporated by reference in their entirety. If the definitions set forth in this section conflict or otherwise contradict the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth in this section shall take precedence over the following definitions incorporated herein by reference.

[0014] As used herein, "a" or "an" means "at least one" or "one or more."

[0015] As used herein, the term "about," when used to describe a numerical value, encompasses a range of up to ±15% of that numerical value, unless the context clearly dictates otherwise.

[0016] Although compositions and methods are described in terms of "comprising" (which may be interpreted as "including but not limited to") various components or steps, compositions and methods may also "consist essentially of" or "consist of" various components and steps, and such terms should be interpreted as defining an essentially closed group of members.

[0017] As used herein, "biological sample" refers to hematological, cytological, and histological specimens, such as cells, cell cultures, single-cell organisms (e.g., yeast and bacteria), 3D cell cultures (e.g., spheroids and organoids), tissues, whole organisms (e.g., flies or worms), cell-free extracts, or liquid samples (e.g., blood, serum, plasma, or saliva). Biological samples may refer to samples that have been processed by filtration and / or centrifugation, and may include cell culture supernatants and homogenized tissues or pulverized cells. Tissue specimens may be any type of nervous, epithelial, muscle, or connective tissue, including organ tissues. Tissue specimens may be fresh or frozen, fixed, or preserved using common histological techniques. Biological samples may be from plants or animals (e.g., humans, mice, flies, worms, fish, frogs, fungi, etc.).

[0018] As used herein, "purifying" or "purification" refers to the preparation of a peptide, polypeptide, or protein in pure or substantially pure form. Purification also refers to the removal of contaminants from a sample, including contaminants originating from the sample or introduced during sample handling. In particular, formulations and methods for the removal of hydrophobic, hydrophilic, and cationic contaminants from biological samples are provided herein. A peptide or polypeptide sample is considered pure or substantially pure when non-peptide and non-polypeptide compounds are essentially removed (i.e., peptides are the only analytes above the level of detection) as measured by mass spectrometry and / or UV / vis spectroscopy. A sample is considered pure or substantially pure when the level of purified peptides and polypeptides in the sample is at least 70%, more preferably at least 80%. In some embodiments, the level of purified peptides and polypeptides is greater than about 80%, or greater than about 90%, or greater than about 95%. In some embodiments, the sample is free of contaminants (i.e., the level of purified peptides and polypeptides is 100%). If the sample is in liquid form, the level of purity is assessed in terms of weight / volume (w / v) percentage. If the sample is in solid (e.g., lyophilized) form, the level of purity is assessed in terms of weight / weight (w / w) percentage.

[0019] As used herein, "peptide" and "polypeptide" are used interchangeably to refer to a polymeric chain formed from two or more amino acid residues, where pairs of amino acid residues are covalently linked through peptide bonds. As used herein, a peptide contains 2 to 50 amino acids. Peptides may include post-translational or other types of modifications, including, but not limited to, phosphorylation, glycation, glycosylation, and methylation. As used herein, "peptide" may refer to a single peptide compound or a mixture of two or more peptide compounds. As used herein, a "peptide" may be chemically synthesized by the condensation reaction of the carboxyl group of one amino acid with the amino group of another amino acid. As used herein, a "peptide" may also be generated by digestion of a protein using a proteolytic enzyme (e.g., trypsin, Lys-C, AspN, or GluC) or by a chemical reaction that selectively hydrolyzes protein amide bonds. A peptide may also have one or more charged or potentially charged groups (depending on pH) at the N-terminus, C-terminus, and / or side chains. Upon digestion with proteolytic enzymes, proteins produce peptides that carry at least one charge, and peptides often carry at least two positive charges. Because peptides contain one or more charges or potentially charged groups at the N-terminus, C-terminus, and / or side chains, one or more cations and / or one or more anions typically accompany peptides or polypeptides to keep the compound electrostatically neutral. Representative examples of counterions include alkali metal ions, alkaline earth metal ions, halides, sulfates, carbonates, phosphates, and acetates.

[0020] As used herein, the terms "mass tag" and "tandem mass tag" are used interchangeably to refer to chemical labeling reagents containing one or more stable isotopes that can be identified by mass spectrometry. "Mass tag" can also refer to a series of reagents that have the same chemical structure but different numbers of stable isotopes or isotopologues. "Mass tag" can also refer to a series of reagents that have the same chemical structure and the same number of stable isotopes but different distributions within the chemical structure, which can be distinguished by mass after gas-phase fragmentation in a mass spectrometer. As used herein, "mass tag" also refers to a series of reagents that can be used to covalently label molecules in two or more samples. Two or more samples can be combined into one sample for mass spectrometry analysis. In some embodiments, "mass tags" can be used to covalently label nucleic acids, peptides, carbohydrates, lipids, or other small molecules derived from biological samples.

[0021] Methods, formulations, and kits are described for purifying peptides and proteolytic digests from biological samples, such as tissues, cells, and biological fluids. Generally, methods and formulations are described herein for removing contaminants from peptides derived from biological samples. Samples derived from biological specimens can contain a variety of hydrophobic, hydrophilic, and ionic contaminants. The contaminants can originate from the biological sample itself or can be introduced during processing of the biological sample. The described methods implement a series of washing steps to remove undesired contaminants from the sample. A particular advantage of the methods and associated washing solutions disclosed herein over known purification methods is that both hydrophobic and hydrophilic monovalent cationic contaminants (regardless of origin) can be removed from the sample. Thus, highly pure peptides that are substantially free of contaminants can be provided. The isolated peptides are suitable for use in demanding downstream applications and assays (e.g., LC-MS) that require extremely pure samples. Removal of unwanted debris and components (e.g., salts, fats, lipids, sugars, etc.) from biological samples can prevent instrument damage and improve the quality of MS proteomics data (e.g., by removing undesired ions). For example, when used in LC-MS applications, peptides purified according to the methods of the present invention can minimize chromatographic interference, ionization suppression, and clogging of liquid chromatography (LC) piping components. Furthermore, removal of contaminants can improve LC-MS peptide and protein identification rates through reduced sample complexity in the gas phase, with fewer charge states (e.g., M+, M+2, M+3, etc.) and / or charge species (e.g., M+Na, M+2Na, etc.). Improved LC-MS spectral quality can also improve peptide and protein identification rates during data analysis using database searching, spectral library matching, or de novo sequencing.

[0022] The described methods and formulations offer improvements over existing purification methods that employ standard reverse-phase and cation exchange chromatography resins. As discussed above, a drawback of reverse-phase desalting methods is that hydrophobic molecules, such as lipids and detergents, can remain bound to the resin through hydrophobic interactions. When eluting bound peptides from the resin using an acidic solution containing an organic solvent, hydrophobic molecules can also be eluted, thereby contaminating the peptide sample. To address some of the shortcomings associated with the use of reverse-phase resins for the purification of peptides from biological samples, strong cation exchange resins can be used as an alternative to the reverse-phase approach for peptide sample cleanup.

[0023] In one typical method using a cation exchange resin, a sample is acidified and loaded onto the cation exchange resin in aqueous solution. Hydrophilic, anionic, and neutral species pass through the resin, while all cationic and hydrophobic species remain bound to the resin via ion exchange or hydrophobic interactions, respectively. The resin is then washed with a first acidic aqueous solution of low ionic strength (e.g., 0.1% formic acid in water) to remove any excess hydrophilic cations from the resin matrix. The resin is then washed with a solution of the same ionic strength and acidity but containing an organic solvent component (e.g., 0.1% formic acid in 70% acetonitrile). A second wash solution removes neutral and anionic hydrophobic species (e.g., lipids, surfactants, etc.) that are bound to the resin via hydrophobic interactions. After treatment with the second wash solution, only cationic species from the sample remain bound to the resin (e.g., atomic cations such as sodium, various small and large amines, and peptides). The peptides are then eluted with a basic solution containing an organic solvent component to ionize the carboxylic acid groups on the peptides and eliminate any hydrophobic interactions at a high pH, ​​which reduces the affinity of the amino groups for the resin. Furthermore, the ionic strength of the elution solution is preferably sufficient (e.g., 0.1% (v / v) or greater) to compete with the peptide amino groups for cation exchange binding sites. A typical elution solution may contain a combination of ammonia or trimethylamine (TEA) in 30% or greater acetonitrile (ACN) organic solvent.

[0024] It is important to note here that all peptides generated from proteolytic digestion with trypsin contain at least two positive charges at the N-terminus and at the C-terminal lysine / arginine residues at low pH. While both monovalent and divalent cations can remain bound to cation exchange resins, divalent cationic peptides have a higher affinity for strong cation exchange resins than cationic species with a single positive charge. As a result, unwanted non-peptide cations are eluted from the resin along with the peptides. To address this issue, improved methods and wash solutions are described herein that remove non-peptide singly charged cationic species from the resin while simultaneously removing neutral and anionic hydrophobic species (e.g., lipids, surfactants, etc.) that are bound to the resin through hydrophobic interactions. Unexpectedly, by optimizing the ionic strength of the wash solution using various formulations of volatile salts under acidic pH conditions, it was possible to simultaneously maximize the removal of unwanted cations while maintaining peptide retention on the resin. Thus, the described formulations and methods offer a significant improvement over existing methods for purifying peptides from biological samples.

[0025] In one aspect, a method for isolating highly pure peptides from a biological sample is provided, wherein the isolated peptides are substantially free of contaminants. The method includes a series of washing steps to remove undesired species that may contaminate the purified peptide sample. Resin washing refers to a procedure in which the stationary phase resin or other solid material used for chromatography is contacted with liquid mobile phase components. Washing of the chromatographic support can be achieved by incubating and / or mixing the resin with a wash buffer. Washing can also be achieved by passing a wash buffer through the resin in a column format using centrifugation, vacuum, gravity, positive pressure, or other means. A particular advantage of the method disclosed herein over known purification methods is that both hydrophobic and hydrophilic monocationic contaminants can be removed from the peptide sample. The purified peptides are sufficiently pure for interrogation in further downstream assays and applications that benefit from extremely pure samples. Examples of downstream applications include, but are not limited to, Western blotting, immunoprecipitation / purification, ligand-receptor binding assays, NMR spectroscopy, colorimetric and fluorometric qualitative and quantitative assays, and LC-MS analysis. For example, it has been found that the purity of peptides resulting from the improved process can significantly enhance sample quality metrics associated with LC-MS analysis (e.g., peptide and protein identification, sampling success rate, etc.).

[0026] The methods described herein offer particular advantages in the context of purifying samples containing peptides labeled with mass tags for comparative (i.e., relative quantification) analysis of peptide and protein samples. For example, peptide samples (either before or after cleanup) can be labeled at the N-terminus and lysine residue side chains with amine-reactive Tandem Mass Tag (TMT) reagents. TMT reagents are monovalent cations that are nonvolatile at low pH. However, excess TMT reagent in a sample can interfere with colorimetric and MS-based assays. The methods disclosed herein provide for the removal of excess TMT reagent from the sample. Removal of the TMT reagent results in a significant reduction in spectral interference in MS-based analysis and in quantitative colorimetric assay measurements of sample peptide yields, resulting in improved peptide and protein identifications.

[0027] A biological sample may contain peptides (e.g., peptides resulting from digestion of proteins or polypeptides) as well as residual components from the biological sample and / or species remaining from sample processing. Contaminants may include hydrophilic and / or hydrophobic species as well as charged species. For example, a biological sample may be contaminated with salts, detergents, lipids, nucleic acids, and other types of small molecules (e.g., carbohydrates, metabolites, nucleotides, etc.). A sample may contain cationic and / or anionic species. In some embodiments, a sample may contain monovalent and / or divalent cationic species. In other embodiments, a sample contains hydrophilic contaminants (e.g., neutral or ionic species) in addition to monovalent and / or divalent cationic species. Examples of hydrophilic contaminants include, but are not limited to, salts, nucleic acids, and carbohydrates. Representative examples of monovalent cationic contaminants include ammonium, sodium, potassium, tris(hydroxymethyl)aminomethane (TRIS), hydroxylamine, ethanolamine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), and glycine.

[0028] In certain embodiments, protein digests or peptides may be derivatized with mass tags or other labeling reagents, such as amine-, sulfhydryl-, carboxyl-, and carbonyl-reactive Tandem Mass Tag (TMT) reagents (available from Thermo Fisher Scientific, Waltham, MA), iTRAQ reagents (available from SCIEX, Framingham, MA), N,N-dimethylleucine (di-Leu) reagents, combinatorial mass tags, fluorescent dyes, biotinylation reagents, etc. Labeling with mass tags or other labeling reagents may introduce contaminants into the sample, for example, in the form of excess unreacted labeling reagent. For protein digest or peptide samples labeled with mass tags (e.g., TMT) or other labeling reagents, the sample prior to purification may contain residual mass tags and components (e.g., buffer salts) used in the labeling and quenching reactions. The contaminants may be unreacted mass tags or labels. Alternatively, the contaminants may be formed during the labeling reaction. In some embodiments, contaminants are the derivatives of label or mass tag that form during the process of labeling peptide or protein digest.In some embodiments, mass tag derivatives can be in the form of monovalent cations.For example, the monovalent cationic derivatives of mass tag such as TMT can include, for example, the NHS ester of unreacted TMT reagent, the acid derivative of TMT reagent that forms by hydrolysis of mass tag, and the amide derivative of TMT that forms during the reaction of mass tag with amine or amine-containing compound.

[0029] One embodiment of the method is used to purify biological samples, such as peptides or protein digests, by washing the sample bound to a purification resin with a single solution to remove monovalent cationic and hydrophobic contaminants. The processed biological sample may contain hydrophobic components, such as exogenously introduced surfactants, which aid in protein solubilization, as well as buffer salts, which are typically introduced to maintain the protein's pH and solubilization properties. After proteolytic digestion of the sample proteins, removal of these contaminants is crucial for achieving optimal analysis by mass spectrometry and other analytical techniques. In one exemplary method, the sample is loaded onto a hydrophobic, polymer-based cation exchange material under acidic conditions (e.g., pH about 1-5). Typically, the cation exchange material is contained within a column to facilitate easy sample handling and processing. Under acidic conditions, the sample can bind to the cation exchange material. In the next step of the process, the cation exchange material is washed with an acidic solution (e.g., pH about 1-5). In certain embodiments, the pH is about 2-4. The acidic solution may contain water and one or more volatile components, such as a volatile organic solvent and a volatile salt. The volatile salt may include a volatile acid (e.g., formic acid, acetic acid, trifluoroacetic acid, or trichloroacetic acid) and a volatile base (e.g., trimethylamine, ammonia, triethylamine, piperidine, or butylamine). Typically, the acidic solution may contain 20% (v / v) or more of a volatile organic solvent (e.g., acetonitrile, methanol, ethanol, n-propanol, isopropanol, or acetone). For example, the acidic solution may contain about 20% to 40%, 40% to 60%, 60% to 80%, or more than 80% (v / v) of a volatile organic solvent. The acidic solution may further include a volatile salt. The amount of volatile salt in the solution may be adjusted to maintain the pH at a desired level. For example, to maintain the pH of the solution at about 1 to 5, the concentration of the volatile salt may range from about 0.01% to about 1.0% (v / v).In some embodiments, the acidic solution contains water, 60% (v / v) or more of a volatile organic solvent (e.g., acetonitrile, methanol, ethanol, n-propanol, isopropanol, or acetone), and about 0.1% to 1.0% (v / v) of a volatile salt (e.g., a salt containing a volatile acid component such as formic acid, acetic acid, and / or trifluoroacetic acid, and a volatile base such as ammonia, triethylamine, and / or piperidine), such that the pH of the solution is maintained at about pH 2 to 4. In some embodiments, one or more volatile acids or volatile bases may be used. Upon treatment with the acidic solution, peptides are retained on the cation exchange material, and monovalent cationic and / or hydrophobic contaminants may be removed from the cation exchange material. The retained peptides may then be eluted from the cation exchange material using an alkaline or neutral solution. The alkaline or neutral solution may include a combination of a volatile organic solvent and water. In certain embodiments, the elution solution contains 20% (v / v) or more of a volatile organic solvent (e.g., 20%-40%, 40%-60%, 60%-80%, or more than 80% (v / v)). Typically, an alkaline or neutral solution has a pH of 5 or greater. In certain embodiments, the solution has a pH greater than 7, greater than 8, greater than 9, greater than 10, or greater than 11. Additionally, the alkaline or neutral solution may contain a volatile base and / or a volatile salt as described herein. The amount of volatile salt in the elution solution may be adjusted to maintain the pH at a desired level. To maintain the pH of the elution solution at 5 or greater, the concentration of the volatile salt may range from about 0.01% to about 1.0% (v / v). In some embodiments, the elution solution comprises 40% (v / v) or more volatile organic solvent (e.g., acetonitrile, methanol, ethanol, n-propanol, isopropanol, or acetone) and about 0.01%-1.0% (v / v) volatile salt (e.g., salts containing volatile acid components such as formic acid, acetic acid, and / or trifluoroacetic acid, and volatile bases such as ammonia, triethylamine, and / or piperidine), such that the pH of the solution is maintained at about pH 8-12.

[0030] One exemplary method provided herein is used to purify biological samples, such as peptides or protein digests. The method involves washing the resin-bound sample with more than one solution to first remove hydrophobic and monocationic contaminants, and then subsequently remove hydrophilic contaminants. For example, the sample may contain high concentrations of preservatives (e.g., carbohydrates), which, if not sufficiently removed, can result in significant levels of interference in downstream analysis of the sample by MS. Efficient removal of such contaminants typically requires the use of wash solutions containing 20% ​​(v / v) or less of a volatile organic solvent. Therefore, for a particular sample, it may be advantageous to include multiple wash solutions to remove hydrophilic, hydrophobic, and ionic contaminants from the sample. In one exemplary method for implementing multiple wash solutions, a sample containing peptides is loaded onto a cation exchange material disclosed herein. The material is then washed with a first acidic solution (pH about 2-4). In some embodiments, the first acidic solution contains 20% (v / v) or less of a volatile organic solvent, water, and a volatile acid. In some embodiments, the first acidic solution contains 10% to 20%, or 5% to 10%, or 1% to 5%, or less than 1% (v / v) of a volatile organic solvent. In some embodiments, the first acidic solution contains less than 5% (v / v) of a volatile organic solvent and about 0.01% to 0.5% (v / v) of a volatile acid (e.g., formic acid, acetic acid, trifluoroacetic acid, or trichloroacetic acid), such that the pH of the solution is maintained at about pH 2 to 4. Treatment with the first acidic solution removes hydrophilic contaminants from the cation exchange material, while leaving the peptides retained on the cation exchange material. The cation exchange material is then washed with a second acidic solution (e.g., about pH 1 to 5) to remove monovalent cationic and / or hydrophobic contaminants from the cation exchange material, while leaving the peptides bound to the cation exchange material.The second acidic solution can contain water and one or more volatile components, such as a volatile organic solvent (e.g., acetonitrile, methanol, ethanol, n-propanol, isopropanol, or acetone) and a volatile salt (e.g., a salt formed from a volatile acid, such as formic acid, and a volatile base, such as trimethylamine). Typically, the second acidic solution contains 20% (v / v) or more of a volatile organic solvent. For example, the acidic solution can contain about 20%-40%, 40%-60%, 60%-80%, or more than 80% (v / v) of a volatile organic solvent. In some embodiments, the acidic solution contains more than 60% (v / v) of a volatile organic solvent and about 0.01%-1.0% (v / v) of a volatile salt, such that the pH of the solution is maintained at about pH 2-4. The retained peptides can be eluted from the cation exchange material using alkaline or neutral solutions as disclosed herein.

[0031] A representative method for isolating pure peptides from a biological sample using the formulations and methods disclosed herein is shown in FIG. 1. Referring to FIG. 1, peptide purification workflow 100 involves digesting a biological sample using methods familiar to those skilled in the art to provide a crude digest sample 110 containing digested biological material (e.g., cells, tissues, nucleic acids, proteins, polypeptides, lipids, carbohydrates, etc.) and one or more contaminants, such as salts, detergents, lipids, and other small neutral molecules. The digested sample 110 is acidified by the addition of a neat or dilute acid solution sufficient to lower the pH to between 1 and 4. The digested sample 110 is then transferred to a hydrophobic, polymer-based cation exchange material 115 (step 1). The cation exchange material may be contained within a cleanup column 116 fitted with an outlet port 117. The column may be contained within a centrifuge tube 118. The centrifuge tube containing the cleanup column may be centrifuged under vacuum to flush components 120 (e.g., digested biological material) from the sample through the resin 115 contained in the column 116. Components 120 with little or no affinity for the cation exchange material 115 flow through the cation exchange material, exit through port 117, and are collected in centrifuge tube 118. A first acidic solution (Wash Solution A) is applied to the cation exchange material and centrifuged under vacuum, thereby washing hydrophilic contaminants (e.g., neutral and anionic components) 130 through the cation exchange material, while cationic and hydrophobic species (e.g., peptides and / or digested proteins) are retained on the cation exchange material 115 (Step 2). The hydrophilic contaminants 130 are discarded. A second acidic solution (Wash Solution B) is applied to the cation exchange material, and the column is centrifuged under vacuum to remove hydrophobic (e.g., neutral, anionic, and monovalent cationic) and hydrophilic monovalent cationic contaminants 140 from the resin 115 (Step 3). Divalent cationic species are retained on the cation exchange material, while monovalent cationic and / or hydrophobic contaminants 140 are removed from the cation exchange material by centrifugation under vacuum.Because peptides produced by enzymatic digestion of polypeptides or proteins have at least two positive charges (i.e., divalent cations), the peptides are retained on the cation exchange material 115 after step 3. The peptides are then eluted from the cation exchange material with an alkaline or neutral solution (step 4) to provide purified peptide solution 150. Purified peptide solution 150 can optionally be dried (e.g., by lyophilization) (step 5) to provide dried peptides 160 that are sufficiently pure for use in further downstream applications.

[0032] One embodiment of the method involves purifying a biological sample, such as a peptide or protein digest, by washing the sample bound to a purification resin with more than one solution to remove hydrophilic contaminants before hydrophobic and monovalent cationic contaminants. In yet another method, a peptide-containing sample is loaded onto a cation exchange material disclosed herein. The cation exchange material is then washed with a first acidic solution, where the first acidic solution comprises more than 20% (v) of a volatile organic solvent, water, and a volatile salt, such that the peptide-containing compound is retained on the cation exchange and the monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material.

[0033] The first acidic solution may contain water and one or more volatile components, such as a volatile organic solvent and a volatile salt. Typically, the first acidic solution contains 20% (v / v) or more of a volatile organic solvent. For example, the acidic solution may contain about 20%-40%, 40%-60%, 60%-80%, or more than 80% (v / v) of a volatile organic solvent. In some embodiments, the acidic solution contains more than 60% (v / v) of a volatile organic solvent and about 0.01%-1.0% (v / v) of a volatile salt, such that the pH of the solution is maintained at about pH 2-4. Next, a second acidic solution is used to wash the cation exchange material. In certain embodiments, the second acidic solution contains 20% (v / v) or less of a volatile organic solvent, water, and a volatile acid. Treatment with the second acidic solution removes hydrophilic contaminants while leaving the peptide on the cation exchange material. In some embodiments, the second acidic solution contains 20% (v / v) or less of a volatile organic solvent, water, and a volatile acid. In some embodiments, the first acidic solution contains 10%-20%, or 5%-10%, or 1%-5%, or less than 1% (v / v) of a volatile organic solvent. In some embodiments, the first and second solutions contain less than 5% (v / v) of a volatile organic solvent and about 0.01%-0.5% (v / v) of a volatile acid, such that the pH of the solution is maintained at about pH 2-4. Retained peptides can be eluted from the cation exchange material using alkaline or neutral solutions as disclosed herein.

[0034] In another embodiment, the methods provided herein further comprise a step of labeling protein digests or peptides with a labeling reagent (e.g., mass tag). The addition of components (e.g., buffer salts, quenching reagents, and residual labeling reagents) to facilitate the labeling of proteins, protein digests, and peptides with mass tags may introduce additional contaminants into biological samples, which may deteriorate the quality of LC-MS data and / or damage the chromatography column and pump. Advantageously, the formulations and methods provided herein can remove contaminants introduced during the labeling process, providing peptides labeled with mass tags or other labeling reagents that are substantially free of such contaminants and suitable for analysis using LC-MS systems. The methods provided herein can remove excess TMT reagent from samples, thus resulting in significantly reduced spectral interference in MS-based analyses, reduced interference in quantitative colorimetric assay measurements of sample peptide yields (since TMT reagent interferes with these types of assays), and reduced physical deposition and accumulation of these materials on LC-MS instrument components, which ultimately extends the high sensitivity of the instrument system and results in increased numbers of peptide and protein identifications.

[0035] Mass tags include tandem mass tag reagents, such as TMT and TMTPRO Label Reagent, commercially available from Thermo Fisher Scientific (Waltham, MA). In certain embodiments, protein digests are labeled with mass tags before peptide cleanup. In other embodiments, peptides can be labeled with TMT reagents after peptide cleanup.

[0036] Thus, methods are provided herein that further include labeling a protein digest or purified peptide with a TMT reagent. A typical method for labeling a protein digest with a TMT reagent includes combining a protein digest dissolved in a slightly basic (e.g., pH 7.5-9) buffer (e.g., 100 mM TEAB or HEPES) solution with a TMT reagent dissolved in an organic solvent (e.g., acetonitrile), incubating the mixture at room temperature, and then quenching the labeling reaction using a solution containing a primary amine (e.g., hydroxylamine).

[0037] A typical method for labeling purified peptides with TMT reagent involves dissolving the dried peptide sample in a slightly basic (e.g., pH 7.5-9) buffered (e.g., 100 mM TEAB or HEPES) solution. The dissolved peptide sample is then combined with TMT reagent dissolved in acetonitrile and incubated at room temperature. The labeling reaction is quenched using a solution containing hydroxylamine. Once quenched, the labeled sample is acidified (pH < 3) using TFA and desalted.

[0038] The methods provided herein practice solid-phase extraction resins. The extraction resin may include a hydrophobic polymer-based cation exchange material. Such materials are well known to those skilled in the art, and a variety of cation exchange resins may be used in the practice of the disclosed methods. Representative examples of suitable hydrophobic polymer-based cation exchange materials include sulfonated divinylbenzene polystyrene, sulfonated polydivinylbenzene resin, sulfonated polydivinylbenzene / polystyrene resin, and sulfonated polydivinylbenzene / polystyrene resin. Representative examples of commercially available hydrophobic polymer-based cation exchange materials include Oasis MCX and POROS XS Strong Cation Exchange Resins, available from Waters Corporation (Milford, MA) and Thermo Fisher Scientific (Bedford, MA), respectively. In some embodiments, the extraction resin may include a combination of cation exchange materials. In certain embodiments, the purification resin is a physical blend of cation exchange materials. In certain embodiments, the extraction resin may be or include a copolymer formed from a combination of a monomer, such as divinylbenzene or styrene, with a pyrrolidone monomer. In some embodiments, the cation exchange material is a copolymer of pyrrolidone and a sulfonated monomer, such as divinylbenzene or sulfonated polydivinylbenzene. In certain embodiments, other types of hydrophobic and / or hydrophilic resins (e.g., C18 resin, non-sulfonated polydivinylbenzene / polystyrene resin, silica, agarose, Sepharose, etc.) with or without strong cation exchange properties can be combined (e.g., as a physical blend) with the hydrophobic polymer-based cation exchange material disclosed herein. In certain embodiments, the extraction resin is a combination of a strong cation exchange material (e.g., a sulfonated monomer disclosed herein) and a hydrophilic and / or hydrophobic resin. In certain embodiments, the binding mode of the hydrophilic and / or hydrophobic resin is the same as that of the strong cation exchange material.

[0039] Solid-phase extraction (SPE) resins can be in the form of magnetic particles (e.g., beads). For example, SPE resins can be composed of a polymer material further comprising a magnetic material. Application of a magnetic field to a sample containing an analyte bound to the magnetic polymer particles allows for isolation of the analyte without the use of centrifugation or filtration. By "magnetic," it is meant herein that the polymer particles contain superparamagnetic crystals. Thus, magnetic polymer particles are magnetically displaceable but not permanently magnetizable. Many processes for preparing magnetic polymer particles are known, many of which involve preparing maghemite or magnetite-containing polymer particles from preformed magnetic iron oxide (e.g., magnetite). Magnetic polymer particles can be implemented in the peptide purification protocols disclosed herein and can be easily automated on a wide range of automation platforms.

[0040] The cleaning solutions provided herein utilize one or more volatile components, such as organic solvents, bases, and salts. Volatile components are particularly useful because they can be easily evaporated under vacuum without introducing additional contaminants into the sample. Volatile components are also useful because they are compatible with electrospray ionization-based mass spectrometry. Representative examples of volatile organic solvents that can be used in the disclosed methods include, but are not limited to, acetonitrile, methanol, ethanol, n-propanol, isopropanol, and acetone. In some cases, combinations of volatile organic solvents can be used in the disclosed methods. Representative examples of volatile salts that can be used in the disclosed methods can include volatile acids and / or volatile bases. Suitable volatile bases include, for example, trimethylamine (TEA), ammonia, piperidine, and butylamine. Suitable volatile acids include, for example, formic acid, acetic acid, trifluoroacetic acid, and trichloroacetic acid. In certain embodiments, volatile salts include volatile bases and volatile acids, such as ammonium acetate, ammonium formate, ammonium trifluoroacetate, triethylammonium formate, triethylammonium acetate, triethylammonium trifluoroacetate, or ammonium bicarbonate.

[0041] Further provided herein is a kit for purifying biological samples containing peptides.The kit can be used to isolate peptides from biological samples such as cells or tissues.Peptides can be produced by digestion of proteins or polypeptides.

[0042] In one aspect, kits are provided for performing efficient and reproducible processing of cells (e.g., cultured mammalian cells), biological fluids (e.g., plasma or serum), and tissues for proteomic MS analysis and other types of assays. The kits may include preformulated buffers, MS-grade enzyme mixes, peptide cleanup (plates and columns), and optimized, time-efficient protocols for generating peptide samples compatible with LC-MS analysis. Purified peptide samples can be prepared in a few hours (e.g., less than 4 hours) using the kits and methods provided herein, thereby significantly reducing the standard processing time associated with existing kits and methods. Protein samples of about 1 μg to about 10 mg or more can be processed using the disclosed methods and kits, providing high yields of purified peptide samples. In some embodiments, the kits can be used to process protein samples ranging from about 0.5 mg to about 2 mg.

[0043] The kits provided herein may include preformulated buffers, MS-grade enzymes (e.g., nucleases, reduction / alkylation solutions for cysteine ​​modification, and trypsin / Lys-C protease mixes for protein digestion), and protocols for generating MS-compatible peptide samples. Additionally, the kits may include peptide cleanup plates or vials and solutions for preparing detergent-free peptide samples for direct LC-MS analysis or further sample processing, such as isobaric tag (e.g., TMT™ Reagent) labeling, phosphopeptide enrichment, or fractionation (e.g., high-pH reverse-phase fractionation). In certain embodiments, the kits provided herein may include one or more wash solutions described herein, as well as one or more additional components, such as a lysis solution, a universal nuclease, a reduction solution, an alkylation solution, an enzyme reconstitution solution, a trypsin / Lys-C protease mix, enzymes, a digestion stop solution, and an elution solution.

[0044] A representative example of a kit for purifying peptides of biological origin includes (a) a hydrophobic polymer-based cation exchange material; (b) an acidic solution containing a volatile salt, water, and more than 20% (v / v) of a volatile organic solvent; (c) a neutral or alkaline solution containing a volatile base, water, and a volatile organic solvent; and (d) instructions for using the kit to purify a biological sample.

[0045] Another example of a kit for purifying peptides originating from a biological sample includes: (a) a hydrophobic polymer-based cation exchange material; (b) an acidic solution comprising more than 20% (v / v) of a volatile organic solvent, a volatile salt, and water; (c) an acidic solution comprising 20% ​​or less (v / v) of a volatile organic solvent, a volatile salt, and water; (d) a neutral or alkaline solution comprising a volatile base, water, and a volatile organic solvent; and (e) instructions for using the kit to purify the biological sample.

[0046] The following examples are included to demonstrate specific embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventor(s) to function well in the practice of the invention and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of this disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the scope of the invention. [Example]

[0047] The examples provided herein utilize the following materials and general methods unless otherwise indicated. Additional materials were obtained from Thermo Fisher Scientific (Rockford, IL) or commercial sources where noted.

[0048] Example 1 Purification of TMT-labeled peptides The following protocol describes a method for purifying peptides labeled with TMT reagent. Peptides can be derived from proteolytic digests of biological samples (e.g., cultured cells, tissues, purified proteins, serum, or plasma), which can be processed as described in Example 3 until dry peptides are obtained after vacuum centrifugation. Alternatively, C18 or another reverse-phase solid-phase extraction cleanup material can be used to desalt the sample before vacuum centrifugation. The dried peptides are dissolved in an appropriate buffer (e.g., 100 mM TEAB pH 8.5 or 100 mM HEPES pH 8.0). Peptides from various samples are labeled with TMT reagent. For a 100 μg peptide sample, label the sample using 0.4–0.8 mg of TMT reagent in 40 μL of acetonitrile for 1 h at room temperature. The reaction is stopped by adding 8 μL of 5% hydroxylamine and incubating for 5 min. Before or after combining the TMT-labeled peptide samples into a single sample, acidify the samples to pH 2–4 using 1–10% formic acid. The combined sample is then loaded onto a cleanup column containing 50-100 mg of the hydrophobic polymeric cation exchange material disclosed herein. Once loaded, the column is centrifuged at 1,000 rpm for 10 minutes. 3 mL of the acidic wash buffer described herein is added, and the column is centrifuged at 2,000 rpm for 2 minutes to remove hydrophilic contaminants (e.g., neutral and anionic components). 3 mL of a second acidic wash buffer described herein is added, and the column is centrifuged at 2,000 rpm for 2 minutes. 3 mL of the second acidic wash buffer is added again, and the column is centrifuged at 2,000 rpm for 2 minutes to remove hydrophobic (e.g., neutral, anionic, and monovalent cationic) and hydrophilic monovalent cationic contaminants. 3 mL of the elution solution described herein is added to the column, and the column is centrifuged at 2,000 rpm for 2 minutes to collect the clean peptide sample. The peptide sample is dried using a vacuum centrifuge. Samples can be resuspended in 100-500 μL of 0.1% formic acid in water for LC-MS analysis.Optionally, peptide yield and concentration may be assessed using a quantitative peptide assay such as the Pierce™ Quantitative Colorimetric Peptide Assay. For LC-MS analysis, 1–10 μg of peptide was adjusted to 0.1–1 μg / μL using a 0.1% formic acid solution in water. Triplicate protein digest samples (1 μg per injection) were separated using a Thermo Scientific™ Dionex™ Ultimate™ 3000 Nano LC system with a 50 cm C18 Thermo Scientific EASY-Spray™ column using an acetonitrile gradient of 3%–28% over 85 minutes, 28%–45% over 30 minutes, at a flow rate of 300 nL / min on a Thermo Scientific™ Fusion™ Trbrid™ mass spectrometer using top-speed data-dependent acquisition. MS spectra were acquired using a resolution of 120K, a target value of 4e5, and a maximum injection time of 50 ms. MS / MS spectra were generated using a CID NCE 35 target value of 1e5 and a maximum injection time of 50 ms. MS / MS / MS spectra were generated using an HCD NCE 60 with a 50K resolution target value of 1e5 and a maximum injection time of 105 ms. LC-MS data were analyzed using the SEQUEST® HT search engine in Thermo Scientific™ Proteome Discoverer™ 2.3 software using static carbamidomethyl (C), dynamic oxidation (M), TMT6plex or TMTpro (K, N-term), and deamidation (N, Q) modifications. Data were searched against the Uniprot human protein database, and results were filtered using a protein FDR threshold of 1% (see Figures 2A-C and 3A-B). Compared with the standard buffer (FA only), the use of an improved acidic wash buffer (TEA and FA) containing volatile salts effectively removed excess quenched and hydrolyzed TMT reagent from the first MS chromatogram.Furthermore, removal of excess TMT reagent was confirmed by a reduction in background signal measured using a colorimetric peptide assay. Removal of the background signal associated with excess TMT reagent is required for accurate peptide measurement for LC-MS analysis.

[0049] Example 2 Purification of TMT Reagent-Labeled Protein Digests from Biological Samples The following protocol describes a method for isolating peptides from biological samples (e.g., cultured cells, tissues, purified proteins, serum, or plasma). Proteins are extracted, reduced, and alkylated from the biological samples using methods well known in the art. The reduced and alkylated proteins are then digested using a mixture of trypsin and Lys-C protease in an appropriate buffer (e.g., 100 mM TEAB pH 8.5 or 100 mM HEPES pH 8.0). Protein digests are from various samples labeled with TMT reagent. For a 10 μg peptide sample, label the sample using 0.04–0.08 mg of TMT reagent in 20 μL of acetonitrile for 15 minutes to 1 hour at room temperature. The reaction is stopped by adding 1–4 μL of 5% hydroxylamine and incubating for 5 minutes. The TMT-labeled peptide samples are acidified to pH 2–4 using 1–10% formic acid before or after combining them into a single sample. The TMT-labeled peptide samples are purified according to the following method. The combined sample is transferred to a dry peptide cleanup column containing a hydrophobic polymeric cation exchange material as disclosed herein. Once loaded, the column is centrifuged at 1,000 rpm for 10 minutes. 300 μL of the acidic wash buffer described herein is added, and the column is centrifuged at 2,000 rpm for 2 minutes to remove hydrophilic contaminants (e.g., neutral and anionic components). 300 μL of a second acidic wash buffer described herein is added, and the column is centrifuged at 2,000 rpm for 2 minutes. 300 μL of the second acidic wash buffer is added again, and the column is centrifuged at 2,000 rpm for 2 minutes to remove hydrophobic (e.g., neutral, anionic, and monovalent cationic) and hydrophilic monovalent cationic contaminants. 300 μL of the elution solution described herein is added to the column, and the column is centrifuged at 2,000 rpm for 2 minutes to collect the clean peptide sample. The peptide sample is dried using a vacuum centrifuge. Samples can be resuspended in 100 μL of 0.1% formic acid in water for LC-MS analysis.Optionally, peptide yield and concentration may be assessed using a quantitative peptide assay such as the Pierce™ Quantitative Colorimetric Peptide Assay. For LC-MS analysis, 1–10 μg of peptide was adjusted to 0.1–1 μg / μL using a 0.1% formic acid solution in water. Triplicate protein digest samples (1 μg per injection) were separated using a Thermo Scientific™ Dionex™ Ultimate™ 3000 Nano LC system with a 50 cm C18 Thermo Scientific EASY-Spray™ column using an acetonitrile gradient of 3%–28% over 85 minutes, 28%–45% over 30 minutes, at a flow rate of 300 nL / min on a Thermo Scientific™ Fusion™ Tribrid™ mass spectrometer using top-speed data-dependent acquisition. MS spectra were acquired using a resolution of 120K, a target value of 4e5, and a maximum injection time of 50 ms. MS / MS spectra were generated using a CID NCE 35 target value of 1e5 and a maximum injection time of 50 ms. MS / MS / MS spectra were generated using an HCD NCE 60 with a 50K resolution target value of 1e5 and a maximum injection time of 105 ms. LC-MS data were analyzed using the SEQUEST® HT search engine in Thermo Scientific™ Proteome Discoverer™ 2.3 software using static carbamidomethyl (C), dynamic oxidation (M), TMT6plex or TMTpro (K, N-term), and deamidation (N, Q) modifications. Data were searched against the Uniprot human protein database, and results were filtered using a protein FDR threshold of 1%. See Figures 4A-4D. Compared with the standard wash buffer, removal of excess TMT reagents and other contaminants using the improved wash buffer reduced the total number of MS / MS scans but increased the total number of peptide spectral matches (PSMs), unique peptides, and protein groups identified by LC-MS.

[0050] Example 3 Peptide purification from biological samples The following protocol describes a method for purifying peptides from proteolytic digests of biological samples (e.g., cultured cells, tissues, purified proteins, serum, or plasma). Proteins are extracted from the biological sample, reduced, and alkylated using methods well known in the art. The reduced and alkylated proteins are then digested using a mixture of trypsin and Lys-C protease. Protein digestion is stopped by adding 1-10% formic acid to a pH range of 2-4. The protein digest sample is transferred to a dry peptide cleanup column containing 5-10 mg of the hydrophobic polymeric cation exchange material disclosed herein. Once loaded, the column is centrifuged at 1,000 rpm for 10 minutes. 300 μL of the acidic wash buffer described herein is added, and the column is centrifuged at 2,000 rpm for 2 minutes to remove hydrophilic contaminants (e.g., neutral and anionic components). Add 300 μL of the second acidic wash buffer described herein and centrifuge the column at 2,000 rpm for 2 minutes. Add 300 μL of the second acidic wash buffer again and centrifuge at 2,000 rpm for 2 minutes to remove hydrophobic (e.g., neutral, anionic, and monovalent cationic) and hydrophilic monovalent cationic contaminants. Add 300 μL of the elution solution described herein to the column and centrifuge at 2,000 rpm for 2 minutes to collect the clean peptide sample. Dry the peptide sample using a vacuum centrifuge. The sample can be resuspended in 100 μL of 0.1% formic acid in water for LC-MS analysis. Optionally, peptide yield and concentration can be assessed using a quantitative peptide assay such as the Pierce™ Quantitative Colorimetric Peptide Assay. For LC-MS analysis, adjust 1-10 µg of peptide to 0.1-1 µg / uL using a 0.1% formic acid solution in water.Triplicate protein digest samples (1 μg per injection) were separated using a Thermo Scientific™ Dionex™ Ultimate™ 3000 Nano LC system with a 50 cm C18 Thermo Scientific EASY-Spray™ column using a Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer with a top-20 data-dependent acquisition method, with an acetonitrile gradient of 3% to 28% over 85 minutes, 28% to 45% over 30 minutes, at a flow rate of 300 nL / min. MS spectra were acquired using a resolution of 70K, a target value of 3e6, and a maximum injection time of 100 ms. MS / MS spectra were generated using a HCD NCE 28 with a resolution of 17.5K, a target value of 1e5, and a maximum injection time of 54 ms. LC-MS data were analyzed using the SEQUEST® HT search engine in Thermo Scientific™ Proteome Discoverer™ 2.3 software, using static carbamidomethyl (C), dynamic oxidation (M), and deamidation (N, Q) modifications. Data were searched against the Uniprot human protein database, and results were filtered using a 1% protein FDR threshold. Figures 5A and 5B show the results of the analysis of peptide and protein identification counts for 10 μg and 100 μg HeLa cell lysate samples prepared using a standard wash solution containing 0.1% formic acid and 70% ACN (Wash B) and an improved wash solution (Wash B+ containing 0.5% formic acid, 0.5% trimethylamine, and 70% ACN). As shown in Figures 5A and 5B, more proteins and peptides were identified using the improved wash solution, which removes additional contaminants, compared to the standard wash solution.

[0051] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following clauses and claims define the scope of the invention, and that methods and structures within the scope of these clauses and claims and their equivalents be covered thereby. Embodiments may follow the following numbered clauses: 1. A method for purifying a biological sample, comprising: (a) contacting a biological sample with a hydrophobic polymer-based cation exchange material under acidic conditions, such that the sample binds to the cation exchange material, and the sample is (i) a peptide, and (ii) contacting the sample with a monovalent cationic contaminant and / or a hydrophobic contaminant; (b) washing the cation exchange material with an acidic solution, the acidic solution comprising greater than 20% (v / v) of a volatile organic solvent, water, and a volatile salt, such that the peptides are retained on the cation exchange material and monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; (c) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile compound selected from a volatile organic solvent, water, and a volatile base, a volatile salt, and combinations thereof. 2. A method for purifying a biological sample, comprising: (a) contacting a biological sample with a hydrophobic polymer-based cation exchange material under acidic conditions, such that the sample binds to the cation exchange material, and the sample is (i) a peptide, (ii) monovalent cationic contaminants and / or hydrophobic contaminants, and (iii) contacting the mixture with a hydrophilic contaminant; (b) washing the cation exchange material with a first acidic solution, the first acidic solution comprising greater than 20% (v / v) of a volatile organic solvent, water, and a volatile salt, such that the peptides are retained on the cation exchange material and monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; (c) washing the cation exchange material with a second acidic solution, the second acidic solution comprising 20% ​​(v / v) or less of a volatile organic solvent, water, and a volatile acid, wherein the peptides are retained on the cation exchange material and hydrophilic contaminants are removed; (d) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile compound selected from a volatile organic solvent, water, and a volatile base, a volatile salt, and combinations thereof. 3. A method for purifying a biological sample, comprising: (a) contacting a biological sample with a hydrophobic polymer-based cation exchange material under acidic conditions, such that the sample binds to the cation exchange material, and the biological sample is (i) a peptide, (ii) monovalent cationic contaminants and / or hydrophobic contaminants, and (iii) contacting the mixture with a hydrophilic contaminant; (b) washing the cation exchange material with a first acidic solution, the first acidic solution comprising 20% ​​(v / v) or less of a volatile organic solvent, water, and a volatile acid, such that the peptides are retained on the cation exchange material and hydrophilic contaminants are removed from the cation exchange material; (c) washing the cation exchange material with a second acidic solution, the second acidic solution comprising greater than 20% (v / v) of a volatile organic solvent, water, and a volatile salt, wherein the peptides are retained on the cation exchange material and monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; (d) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile compound selected from a volatile organic solvent, water, and a volatile base, a volatile salt, and combinations thereof. 4. The method of any one of the preceding clauses, wherein the hydrophilic contaminants are neutral contaminants, anionic contaminants, or a combination thereof. 5. The method of any one of the preceding clauses, wherein the biological sample further comprises a salt, a detergent, a lipid, a small neutral molecule, or a combination thereof. 6. The method of any one of the preceding clauses, comprising contacting the sample with a hydrophobic polymer-based cation exchange material at a pH of 1-5. 7. The method of any one of the preceding clauses, comprising washing the cation exchange material at a pH of 1 to 5. 8. The method of any one of the preceding clauses, comprising eluting the sample from the cation exchange material at a pH above 5 (e.g., 5-11). 9. The method of any one of the preceding clauses, wherein the monovalent cationic contaminant is selected from the group consisting of ammonium, sodium, potassium, tris(hydroxymethyl)aminomethane (tris), hydroxylamine, ethanolamine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), glycine, mass tags, and combinations thereof. 10. The method of any one of the preceding clauses, wherein the monovalent cationic contaminant is or comprises a mass tag or a derivative thereof. 11. The method of any one of the preceding clauses, wherein the hydrophobic contaminant is a lipid or a surfactant. 12. The method of any one of the preceding clauses, wherein the hydrophilic contaminant is a salt, a nucleic acid, or a carbohydrate. 13. The method of any one of the preceding clauses, wherein the biological sample is treated with a proteolytic enzyme (e.g., trypsin or Lys-C) to generate peptides prior to contacting the biological sample with the cation exchange material. 14. A kit for purifying a biological sample containing a peptide, comprising: (a) a hydrophobic polymer-based cation exchange material; (b) an acidic solution containing a volatile salt, water, and more than 20% (v / v) of a volatile organic solvent; (c) a neutral or alkaline solution containing a volatile base, water, and a volatile organic solvent; (d) instructions for using the kit to purify a biological sample. 15. A kit for purifying a biological sample containing a peptide, comprising: (a) a hydrophobic polymer-based cation exchange material; (b) an acidic solution containing more than 20% (v / v) of a volatile organic solvent, a volatile salt, and water; (c) an acidic solution containing 20% ​​(v / v) or less of a volatile organic solvent, a volatile salt, and water; (d) a neutral or alkaline solution containing a volatile base, water, and a volatile organic solvent; (e) instructions for using the kit to purify a biological sample. 16. The method or kit of any one of the preceding clauses, wherein the hydrophobic polymer-based cation exchange material is sulfonated divinylbenzene polystyrene, sulfonated polydivinylbenzene, or sulfonated divinylbenzene / polystyrene / pyrrolidone resin. 17. The method or kit of any one of the preceding clauses, wherein the peptide comprises 2 to 50 amino acid residues. 18. The method or kit of any one of the preceding clauses, wherein the volatile salt comprises a volatile acid and a volatile base. 19. The method or kit of any one of the preceding clauses, wherein the volatile acid is selected from the group consisting of formic acid, acetic acid, trifluoroacetic acid, and trichloroacetic acid. 20. The method or kit of any one of the preceding clauses, wherein the volatile base is selected from the group consisting of trimethylamine, ammonia, triethylamine, piperidine, and butylamine. 21. The method or kit of any one of the preceding clauses, wherein the volatile organic solvent is selected from the group consisting of acetonitrile, methanol, ethanol, n-propanol, isopropanol, acetone, and combinations thereof. 21. The method or kit of any one of the preceding clauses, wherein the sample comprises two or more peptides. 22. The method or kit according to any one of the preceding clauses, wherein the sample is a digested protein or polypeptide. 23. The method of any one of the preceding clauses, comprising eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile organic solvent, water, and a volatile salt. Another aspect of the present invention may be as follows. [1] A method for purifying a biological sample, comprising: (a) contacting the biological sample with a hydrophobic polymer-based cation exchange material under acidic conditions, such that the sample binds to the cation exchange material, and the sample is (i) a peptide, and (ii) contacting the sample with a monovalent cationic contaminant and / or a hydrophobic contaminant; (b) washing the cation exchange material with an acidic solution, the acidic solution comprising greater than 20% (v / v) of a volatile organic solvent, water, and a volatile salt, such that the peptides are retained on the cation exchange material and the monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; (c) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile compound selected from a volatile organic solvent, water, and a volatile base, a volatile salt, and combinations thereof. [2] A method for purifying a biological sample, comprising: (a) contacting the biological sample with a hydrophobic polymer-based cation exchange material under acidic conditions, such that the sample binds to the cation exchange material, and the sample is (i) a peptide, (ii) monovalent cationic contaminants and / or hydrophobic contaminants, and (iii) contacting the mixture with a hydrophilic contaminant; (b) washing the cation exchange material with a first acidic solution, the first acidic solution comprising greater than 20% (v / v) of a volatile organic solvent, water, and a volatile salt, such that the peptides are retained on the cation exchange material and the monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; (c) washing the cation exchange material with a second acidic solution, the second acidic solution comprising up to 20% (v / v) of a volatile organic solvent, water, and a volatile acid, such that the peptides are retained on the cation exchange material and the hydrophilic contaminants are removed; (d) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile compound selected from a volatile organic solvent, water, and a volatile base, a volatile salt, and combinations thereof. [3] A method for purifying a biological sample, comprising: (a) contacting the biological sample with a hydrophobic polymer-based cation exchange material under acidic conditions, such that the sample binds to the cation exchange material, and the biological sample is (i) a peptide, (ii) monovalent cationic contaminants and / or hydrophobic contaminants, and (iii) contacting the mixture with a hydrophilic contaminant; (b) washing the cation exchange material with a first acidic solution, the first acidic solution comprising 20% ​​(v / v) or less of a volatile organic solvent, water, and a volatile acid, such that peptides are retained on the cation exchange material and the hydrophilic contaminants are removed from the cation exchange material; (c) washing the cation exchange material with a second acidic solution, the second acidic solution comprising greater than 20% (v / v) of a volatile organic solvent, water, and a volatile salt, wherein the peptides are retained on the cation exchange material and the monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; (d) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile compound selected from a volatile organic solvent, water, and a volatile base, a volatile salt, and combinations thereof. [4] The method of any one of the preceding aspects, wherein the hydrophilic contaminants are neutral contaminants, anionic contaminants, or a combination thereof. [5] The method of any one of the preceding aspects, wherein the biological sample further comprises a salt, a detergent, a lipid, a small neutral molecule, or a combination thereof. [6] The method of any one of the preceding aspects, comprising contacting the sample with the hydrophobic polymer-based cation exchange material at a pH of 1 to 5. [7] The method of any one of the preceding aspects, comprising washing the cation exchange material at a pH of 1 to 5. [8] The method of any one of the preceding aspects, comprising eluting the sample from the cation exchange material at a pH greater than 5 (e.g., 5 to 11). 9. The method of any one of the preceding aspects, wherein the monovalent cationic contaminant is selected from the group consisting of ammonium, sodium, potassium, tris(hydroxymethyl)aminomethane (tris), hydroxylamine, ethanolamine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), glycine, a mass tag, and combinations thereof. 10. The method of any one of the preceding aspects, wherein the monovalent cationic contaminant is or comprises a mass tag or a derivative thereof.

[11] The method of any one of the preceding aspects, wherein the hydrophobic contaminant is a lipid or a surfactant.

[12] The method of any one of the preceding aspects, wherein the hydrophilic contaminant is a salt, a nucleic acid, or a carbohydrate.

[13] The method of any one of the preceding aspects, wherein the biological sample is treated with a proteolytic enzyme (e.g., trypsin or Lys-C) to generate the peptides prior to contacting the biological sample with the cation exchange material.

[14] A kit for purifying a biological sample containing a peptide, comprising: (a) a hydrophobic polymer-based cation exchange material; (b) an acidic solution containing a volatile salt, water, and more than 20% (v / v) of a volatile organic solvent; (c) a neutral or alkaline solution containing a volatile base, water, and a volatile organic solvent; (d) instructions for using the kit to purify the biological sample.

[15] A kit for purifying a biological sample containing a peptide, comprising: (a) a hydrophobic polymer-based cation exchange material; (b) an acidic solution containing more than 20% (v / v) of a volatile organic solvent, a volatile salt, and water; (c) an acidic solution containing 20% ​​(v / v) or less of a volatile organic solvent, a volatile salt, and water; (d) a neutral or alkaline solution containing a volatile base, water, and a volatile organic solvent; (e) instructions for using the kit to purify the biological sample.

[16] The method or kit of any one of the preceding aspects, wherein the hydrophobic polymer-based cation exchange material is sulfonated divinylbenzene polystyrene, sulfonated polydivinylbenzene, or a sulfonated divinylbenzene / polystyrene / pyrrolidone resin.

[17] The method or kit according to any one of the preceding aspects, wherein the peptide comprises 2 to 50 amino acid residues.

[18] The method or kit of any one of the preceding aspects, wherein the volatile salt comprises a volatile acid and a volatile base.

[19] The method or kit of any one of the preceding aspects, wherein the volatile acid is selected from the group consisting of formic acid, acetic acid, trifluoroacetic acid, and trichloroacetic acid.

[20] The method or kit of any one of the preceding aspects, wherein the volatile base is selected from the group consisting of trimethylamine, ammonia, triethylamine, piperidine, and butylamine.

[21] The method or kit of any one of the preceding aspects, wherein the volatile organic solvent is selected from the group consisting of acetonitrile, methanol, ethanol, n-propanol, isopropanol, acetone, and combinations thereof.

[21] The method or kit of any one of the preceding aspects, wherein the sample comprises two or more peptides.

[22] The method or kit of any one of the preceding aspects, wherein the sample is a digested protein or polypeptide.

[23] The method of any one of the preceding aspects, comprising eluting the retained peptides from the cation exchange material with an alkaline or neutral solution, wherein the alkaline or neutral solution comprises a volatile organic solvent, water, and a volatile salt.

Claims

1. A method for purifying a protein digest sample, comprising: (a) contacting the sample with a hydrophobic polymer-based cation exchange material under acidic conditions; where: As a result of said step, said sample binds to said cation exchange material; The sample is (i) a peptide, (ii) monovalent cationic contaminants and / or hydrophobic contaminants, and (iii) contains hydrophilic contaminants; the monovalent cationic contaminant is or comprises a mass tag or a monovalent cationic derivative thereof; (b) washing the cation exchange material with a first acidic solution; where: the first acidic solution comprises 20% (v / v) or less of a volatile organic solvent, water, and a first volatile acid; the first volatile acid is selected from the group consisting of formic acid, acetic acid, trifluoroacetic acid, and trichloroacetic acid; The result of said step is that peptides are retained on the cation exchange material and that the hydrophilic contaminants are removed from the cation exchange material. (c) washing the cation exchange material with a second acidic solution; where: the second acidic solution comprises greater than 20% (v / v) of a volatile organic solvent, water, and a first volatile salt; the first volatile salt comprises a second volatile acid and a first volatile base; the second volatile acid is selected from the group consisting of formic acid, acetic acid, trifluoroacetic acid, and trichloroacetic acid; the first volatile base is selected from the group consisting of trimethylamine, ammonia, triethylamine, piperidine, and butylamine; the second acidic solution is maintained at a pH of 2 to 4; the peptides are retained on the cation exchange material and the monovalent cationic and / or hydrophobic contaminants are removed from the cation exchange material; and (d) eluting the retained peptides from the cation exchange material with an alkaline or neutral solution; where: the alkaline or neutral solution comprises a volatile organic solvent, water, and a volatile compound selected from a second volatile base, a second volatile salt, and combinations thereof; the second volatile base is selected from the group consisting of trimethylamine, ammonia, triethylamine, piperidine, and butylamine; the second volatile salt is selected from the group consisting of ammonium acetate, ammonium formate, ammonium trifluoroacetate, triethylammonium formate, triethylammonium acetate, triethylammonium trifluoroacetate, and ammonium bicarbonate; A method comprising:

2. The method of claim 1 , wherein the hydrophilic contaminants are neutral contaminants, anionic contaminants, or a combination thereof.

3. The method of any one of claims 1 to 2, comprising contacting the sample with the hydrophobic polymer-based cation exchange material at a pH of 1 to 5.

4. The method of any one of claims 1 to 3, comprising washing the cation exchange material at a pH of 1 to 5.

5. The method of any one of claims 1 to 4, comprising eluting the sample from the cation exchange material at a pH above 5.

6. The method described in claim 1, wherein the hydrophobic contaminant is a lipid or a surfactant.

7. The method of claim 1 , wherein the hydrophilic contaminant is a salt, a nucleic acid, or a carbohydrate.

8. 8. The method of any one of claims 1 to 7, wherein the biological sample is treated with a proteolytic enzyme to generate the peptides prior to contacting the protein digest sample with the cation exchange material.

9. A kit for carrying out the method of claim 1, comprising: (a) a hydrophobic polymer-based cation exchange material; (b) a first acidic solution according to claim 1; and (c) a second acidic solution according to claim 1; and (d) the alkaline or neutral solution of claim 1; (e) instructions for using the kit to practice the method.

10. 9. The method of any one of claims 1 to 8, wherein the hydrophobic polymer-based cation exchange material is sulfonated divinylbenzene polystyrene, sulfonated polydivinylbenzene, or sulfonated divinylbenzene / polystyrene / pyrrolidone resin.

11. 10. The kit of claim 9, wherein the hydrophobic polymer-based cation exchange material is sulfonated divinylbenzene polystyrene, sulfonated polydivinylbenzene, or sulfonated divinylbenzene / polystyrene / pyrrolidone resin.

12. The method of any one of claims 1 to 8 and 10, wherein the peptide comprises 2 to 50 amino acid residues.

13. The kit according to any one of claims 9 and 11, wherein the peptide comprises 2 to 50 amino acid residues.

14. 13. The method of any one of claims 1 to 8, 10 and 12, wherein the volatile organic solvent is selected from the group consisting of acetonitrile, methanol, ethanol, n-propanol, isopropanol, acetone, and combinations thereof.

15. 14. The kit of any one of claims 9, 11 and 13, wherein the volatile organic solvent is selected from the group consisting of acetonitrile, methanol, ethanol, n-propanol, isopropanol, acetone, and combinations thereof.

16. The method of any one of claims 1 to 8, 10, 12 and 14, wherein the sample comprises two or more peptides.

17. 16. The kit of any one of claims 9, 11, 13 and 15, wherein the sample comprises two or more peptides.

18. The method of any one of claims 1 to 8, 10, 12, 14 and 16, wherein the sample is a digested protein or polypeptide.

19. 18. The kit of any one of claims 9, 11, 13, 15 and 17, wherein the sample is a digested protein or polypeptide.

20. 19. The method of any one of claims 1 to 8, 10, 12, 14, 16 and 18, wherein the alkaline or neutral solution comprises a volatile organic solvent, water, and a second volatile salt.

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