Charged quality marking system

Abstract

The present invention generally relates to charged mass label compositions and methods of using the same for detecting target analytes in samples. In certain aspects, the present invention provides charged mass label compositions comprising an affinity reagent and a mass label precursor bound to the affinity reagent. The mass label precursor comprises a label binding unit and a mass label. The label binding unit reversibly binds the mass label to the affinity reagent. The mass label comprises a charge unit and a mass label unit having a predefined mass-to-charge ratio value in a mass spectrum.

Description

[0001] Related Applications

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 62 / 286,115, filed on January 22, 2016, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to charged mass label compositions and methods of using the same for detecting a target analyte in a sample. Background Art

[0004] Some assays have been developed that focus on analyzing rare molecules (e.g., proteins, nucleic acids, etc.) from body fluids for abnormalities, leading to early diagnosis of certain conditions such as cancer. However, in typical body fluid samples, most rare molecules are degraded, and any rare molecules that contain an abnormal change of interest are present in small amounts (e.g., less than 1%) relative to the total amount of molecules in the sample. This results in the inability to detect small amounts of abnormal rare molecules.

[0005] Affinity assays, nucleic acid assays, and cell analysis assays have been developed for detecting rare molecules in samples. Each has some disadvantages. For example, rare molecules of 1 to 50,000 copies per milliliter (femtomoles (fM) or less) cannot be detected by conventional affinity assays, which require a molecule copy number far in excess of the number of rare molecules. Immunoassays are limited by the affinity binding constant of antibodies, which is typically no greater than 10 -12 (1pM). Since the dissociation rate is 10 -13 , so immunoassays require at least a 100-fold excess of antibody, and the solubility of the antibody protein limits the completion of the reaction.

[0006] For nucleic acids, amplification-based methods (e.g., polymerase chain reaction (PCR), digital PCR, quantitative PCR) have been used previously to try to detect these anomalies. However, due to the random nature of the amplification reaction, the molecular population present in small quantities in the sample is usually ignored. In fact, if rare nucleic acids are not amplified in the first few rounds of amplification, it will become increasingly unlikely that rare species will be detected.

[0007] In the case where rare molecules are bound to or contained in cells, cell analysis techniques are used, and target cells containing rare molecules need to be separated. Those cells containing rare molecules usually represent only a small part of the sample in the analysis. Therefore, even before detecting rare molecules in these cells, methods with high separation efficiency and cell recovery are required. After purification, rare cells can be analyzed to the single cell level by conventional scanning microscopes. However, even if scanning and analysis are automated, microscopy methods may take 24 hours or more for each sample to be scanned. In addition, all rare cells with multiple images must be visually inspected by a pathologist to determine the significance of the measured protein amount.

[0008] Attempts have been made to use mass spectrometry (MS) to detect rare events. However, some problems have arisen. For example, the mark of interest can not be separated from sample interference (matrix overlap). The higher detection limit (picomolar (pM) increases to nanomolar (nM)) due to the background in clinical samples is a problem. Since samples less than 1 microliter (jal) are inefficiently captured for ionization and inefficiently separated from complex samples such as blood, it is impossible to use a small nL sample volume. In addition, due to competitive ionization with other ions of the same mass or due to the presence of a specific matrix component, ionization is suppressed, so MS can not detect certain masses usually. These problems will usually cause problems due to false positive and / or false negative results.

[0009] To address these issues, techniques have been developed that rely on the use of mass tags that bind to rare molecules in a sample. This approach allows mass spectrometry to be used for rare event detection because the use of mass tags overcomes the above-mentioned problems associated with directly detecting rare molecules in a sample.

[0010] However, mass tags remain problematic, which limits the full potential of mass spectrometry for rare molecule detection. For example, current MS tags typically utilize peptides bound to affinity agents via reversible disulfide bonds. However, peptides tend to easily exchange protons in the mass spectrometer and typically have low ionization efficiency. Additionally, the chemicals used to release peptides from disulfide bonds, such as TCEP and DTT, may suppress signal intensity in the mass spectrum from the mass tag. While strong acids such as TFA can increase ionization efficiency, they prevent TCEP and DTT from cleaving disulfide bonds, thus requiring a separate liquid to release and ionize the mass tag via spray ionization.

[0011] Thus, the application of mass tag technology remains problematic; namely, mass tags such as peptides do not have ideal ionization efficiency, current mass tags may not fragment into predictable masses that can be separated from background noise, and mass tags are not easily cleaved from affinity reagents. Summary of the invention

[0012] The present invention generally relates to charged mass marker compositions that are designed to be separated from the background mass analysis of a sample and to be ionized efficiently. The compositions of the present invention include an affinity reagent and a mass marker precursor that is bound to the affinity reagent. Due to the specificity of the affinity reagent, each MS marker can correspond to one or more target rare molecules from a sample. Multistage MS (also known as MS n , CID) can be used to detect different rare molecule populations in a sample. The bound affinity agent is exposed to a substance to chemically release the MS label for MS analysis to determine the presence and / or amount of each different MS label. The presence and / or amount of each different MS label is related to the presence and / or amount of each different rare molecule population in the sample.

[0013] The mass label precursor comprises a label binding unit and a mass label. The label binding unit reversibly combines the mass label with an affinity reagent. The reversible combination of the label binding unit allows the MS label to be easily and reliably cleaved from the affinity reagent. The mass label comprises a charge unit (e.g., pre-charged (ionic functional group), such as a quaternary ammonium (quaternary amine) group, or a similar sulfonium or phosphonium cation, or a pyridinium or imidazolium). This charge unit is an ionic type, and is therefore easily observed as a gas phase ion in MS. The mass label also comprises a mass label unit, which has a predefined mass-to-charge ratio value in a mass spectrum. The mass label can produce at least one unique mass fragment due to the fragmentation during ionization, and becomes at least one predictable m / z separated from the background through MS / MS. Because the mass label produces a predictable m / z separated from the background, the characteristic mass label can be easily detected by mass spectrometry.

[0014] Many different types of affinity reagents can be used in the compositions of the present invention. In certain embodiments, the affinity reagent comprises at least one member of a specific binding pair. The affinity reagent can be non-granular or granular and is connected to a mass spectrometry (MS) marker precursor from which the entity comprising the MS marker can be released. In certain embodiments, the mass marker unit and the charge unit are bound to each other by one or more anti-fragmentation bonds when ionized. Typically, the labeling unit comprises a reversible bond, such as an acetal or ketal bond.

[0015] The charge unit includes one or more chemical moieties that are arranged to have a charge when ionized. For example, the charge unit may include one or more chemical moieties selected from the group consisting of Formulas I-VII:

[0016]

[0017] In certain embodiments, at least one X is a quaternary ammonium cation or a quaternary phosphonium cation, which is optionally substituted with at least one R. The R can be selected from the group consisting of hydrogen, an alkyl group, or an aromatic organic derivative. In other embodiments, the charge unit is a chemical moiety selected from the group consisting of carnitine, imidazolium, pyridinium, tetraethylamine, benzalkonium amine, alkylbenzeneethylammonium amine, triphenylphosphonium cation, and trialkyl (2,3-dihydroxypropyl).

[0018] The mass tag unit may include an arrangement of chemical moieties that upon ionization produces one or more fragments. In these embodiments, at least one of the one or more fragments has a predefined mass-to-charge value in the mass spectrum. In certain embodiments, the predefined mass-to-charge value in the mass spectrum of the mass tag or one or more fragments of the mass tag is different from the background mass-to-charge value in the mass spectrum.

[0019] Other aspects of the present invention include methods for detecting a target analyte in a sample. These methods may include introducing a charged mass label composition into a sample containing a target analyte, wherein the charged mass label composition includes an affinity reagent; and a mass label precursor bound to the affinity reagent, wherein the mass label precursor includes a label binding unit and a mass label, the label binding unit reversibly binds the mass label to the affinity reagent, and wherein the mass label includes a charge unit and a mass label unit having a predefined mass-to-charge ratio value in a mass spectrum. The method then includes incubating the sample and the charged mass label composition so that the charged mass label composition binds to the target analyte through an interaction between the affinity reagent of the charged mass label composition and the target analyte. The mass label is then released from the affinity reagent by breaking one or more bonds of the label binding unit. The mass label is ionized and the ionized mass label and / or its fragments are detected in a mass spectrometer, thereby detecting the target analyte in the sample. The method of the present invention may also include quantifying the ionized mass label and / or its fragments, thereby quantifying the target analyte in the sample.

[0020] The methods of the present invention can be used with various types of samples, such as biological samples, environmental samples, agricultural samples, etc. In certain embodiments, the target analyte is a target biomolecule. Typically, the presence of the target biomolecule indicates a disease, such as cancer.

[0021] Other examples described herein relate to methods for detecting one or more different target rare molecule groups in a sample suspected of containing one or more different rare molecules and non-rare molecule groups by mass spectrometry (MS). The concentration of one or more different target rare molecule groups detected with an affinity agent exceeds the concentration of the non-rare molecule, and the affinity agent comprises a specific binding partner of a target rare molecule group for one of the rare molecule groups present. Affinity reagents can be non-granular or granular and are connected with a mass label precursor, which releases a mass label after being exposed to a liquid containing reagent, and the reagent promotes the bond rupture between the mass label in the label precursor and the label binding unit. Four levels of mass labels are carried out to MS analysis to determine the presence and / or amount of each different four levels of mass labels. The presence and / or amount of the four levels of mass labels are relevant to the presence and / or amount of the target rare molecule in the sample.

[0022] Another example described herein relates to a method for detecting one or more different target rare molecule groups in a sample suspected of containing one or more different rare molecules and non-rare molecule groups by mass spectrometry (MS). By combining an affinity reagent connected to a mass marker precursor with the target rare molecule, the concentration of one or more different target rare molecule groups is detected to exceed the concentration of non-rare molecules. After the affinity reagent is combined with the rare molecule, the mass marker is chemically released with a release reagent, and MS analysis is performed to determine the presence and / or amount of a quadruple mass marker, thereby determining the rare molecule. The presence and / or amount of the mass marker is directly related to the presence and / or amount of the target rare molecule in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a diagram of one embodiment of the present invention. Rare molecules / cells are bound to an affinity reagent, which may be particulate or non-particulate. The affinity reagent is in turn functionalized with a label binding unit, which facilitates reversible conjugation with a mass label. The mass label and the label binding unit constitute a mass label precursor. The mass label is composed of a charge unit (which carries a formal charge, such as a quaternary ammonium, sulfonium or phosphonium salt) and a variable mass unit, which is formally neutral and is varied to produce a mass label that appears at a predefined m / z value in a mass spectrum.

[0024] Figure 2 is a charge unit wherein at least one X is a NR moiety (eg, a quaternary ammonium cation), or wherein at least one X is a quaternary phosphonium cation wherein X=PR, wherein R can be hydrogen, an alkyl group, or an aromatic organic derivative such as methyl, benzyl, or phenyl.

[0025] Figure 3 Panels AD show a variable mass unit represented by Y, which comprises at least one atom, such as CH, CH2, O, N, S, P or a molecule with a mass less than 3 kDA, such as phenyl as shown in panels B and D. The variable mass unit Y and the charge unit as part of the mass tag are linked to the affinity reagent or rare molecule represented by R1 through an acetal or ketal bond as shown in panels A and B, or through an imine bond as shown in panels C and D.

[0026] Figure 4 It's L-carnitine.

[0027] Figure 5 Mass tags with imidazolium (m / z 217) and pyridinium (m / z 214) structures as charge units and their respective product ions obtained by MS / MS are shown. In this case, the variable mass unit is composed of all other atoms in the molecule that are not directly connected to the pre-charged atom. The mass tags will produce unique fragment ions when subjected to MS / MS. For the imidazolium mass tag, a unique fragment can be observed at m / z 83 in the product ion MS / MS spectrum. For the pyridinium mass tag, a unique fragment ion can be observed at m / z 80 in the product ion MS / MS spectrum.

[0028] Figure 6 Shows how the affinity reagent or protomolecule represented by R is linked via an acetal or ketal bond to the quaternary mass tag by reaction of a diol with an aldehyde on mass structure Y. The diol is attached to the amine of the affinity reagent or protomolecule by first forming an amide bond via reaction of β-hydroxy-γ-butyrolactone.

[0029] Figure 7 The concentration of mass marker released into the release solution is shown using a calibration curve. The samples (triangles) were diluted four orders of magnitude. The supernatant of the final wash before release was analyzed after two orders of magnitude dilution (squares).

[0030] Figure 8 The mass signatures of higher mass analogs derived from N-substituted imidazoles are shown. In these cases, the charge unit is still the quaternary nitrogen, but the variable mass unit has been changed to produce mass signatures with different masses.

[0031] Fig. 9 A method for predicting the fragmentation of peptide mass tags in a mass spectrometer is described.

[0032] Fig.10 is a picture showing various components and their arrangement in a micro mass spectrometer. DETAILED DESCRIPTION

[0033] The present invention generally relates to charged mass label compositions and methods of using the same for detecting target analytes in samples. In certain aspects, the present invention provides charged mass label compositions comprising an affinity reagent and a mass label precursor bound to the affinity reagent. The mass label precursor comprises a label binding unit and a mass label. The label binding unit reversibly binds the mass label to the affinity reagent. The mass label comprises a charge unit and a mass label unit having a predefined mass-to-charge ratio value in a mass spectrum.

[0034] When subjected to MS / MS, the mass marker produces at least one unique fragment. This fragmentation is predictable and produces at least one mass that can be separated from the background spectral analysis. The mass marker ion can be generated by an ionization event in the surrounding environment ( Figure 1 ). The mass marker and its product ions are detected in the vacuum of a mass spectrometer ( Figure 1 ). A mass tag (eg, a quaternary mass tag) can be attached to an affinity reagent or rare molecule via a tag binding unit (eg, an acetal or ketal bond) and used in methods for detecting rare molecules.

[0035] Some examples relate to methods for detecting one or more different target rare molecule groups in a sample suspected of containing one or more different rare molecules and non-rare molecule groups by mass spectrometry (MS). By binding the composition of the present invention to the target rare molecule in the sample, it is possible to detect that the concentration of one or more different target rare molecule groups exceeds the concentration of non-rare molecules. Attachment can be performed between rare molecules and mass marker precursors with non-particle or particle binding, and after exposure to a liquid containing a change agent, the mass marker is released from the rare molecule. MS analysis can be performed on the treated sample to determine or quantify the presence and / or amount of a quadruple mass marker. The presence and / or amount of a mass marker can be related to the presence and / or amount of a target rare molecule in the sample.

[0036] The term "affinity agent" refers to a member of a specific binding pair. Examples include immune pairs, such as antigen-antibody or hapten-antibody, biotin-avidin pairs, hormone-hormone receptors, enzyme-substrate, nucleic acid duplexes, IgG-protein A, protein-nucleotide pairs and polynucleotide pairs, such as DNA-DNA, DNA-RNA. For example, a nucleic acid (e.g., a polynucleotide) can be an "affinity agent" complementary to a target nucleic acid. A polynucleotide refers to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) of any length or its analogs. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after polymer assembly. The nucleotide sequence may be interspersed with non-nucleotide components. The polynucleotide may be further modified, for example by conjugation with a labeling component. Compositions with affinity may include particles for amplification and capture, which may be entities of organic or inorganic, magnetic or non-magnetic material.

[0037] In certain embodiments, the affinity reagent is a nucleic acid. In certain embodiments, the sequence of the target rare nucleic acid can be determined by reference. For example, mutations that cause cancer due to loss of genetic material and their positions within genes are known in the art. See, for example, Hesketh, The Oncogene Facts Book, Academic Press Limited (1995). Knowing the position of mutations and mutations, those skilled in the art can easily design nucleic acid affinity reagents that will bind to the rare nucleic acid sequences in the sample.

[0038] Alternatively, a sample from a subject can be obtained and sequenced to determine the sequence of rare nucleic acids in the sample. Typically, a sample is obtained from a subject. The sample can be obtained in any clinically acceptable manner, and nucleic acids are extracted from the sample by methods known in the art. Typically, nucleic acids can be extracted from biological samples by a variety of techniques, such as those described by Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, pp. 280-281, 1982), the contents of which are incorporated herein by reference in their entirety.

[0039] Once obtained, the nucleic acid molecules are sequenced by any method known in the art, such as overall sequencing or single molecule sequencing. A conventional method for sequencing is by chain termination and gel separation, as described by Sanger et al., Proc Natl Acad Sci USA, 74 (12): 5463 67 (1977). Another conventional sequencing method involves chemical degradation of nucleic acid fragments. See Maxam et al., Proc. Natl. Acad. Sci., 74: 560 564 (1977). Finally, methods based on hybridization sequencing have been developed. See, for example, Drmanac et al., (Nature Biotech., 16: 54 58, 1998). The contents of each reference are incorporated herein by reference in their entirety.

[0040] In certain embodiments, sequencing is performed by Sanger sequencing technology. Classical Sanger sequencing relates to single-stranded DNA template, DNA primer, DNA polymerase, radioactive or fluorescently labeled nucleotides and modified nucleotides that terminate DNA chain extension. If the mark is not attached to dideoxynucleotide terminator (e.g., labeled primer), or is a monochromatic label (e.g., radioisotope), then the DNA sample is divided into four independent sequencing reactions, which contain four kinds of standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and DNA polymerase. Only one kind (ddATP, ddGTP, ddCTP or ddTTP) of four kinds of dideoxynucleotides is added to each reaction. These dideoxynucleotides are chain termination nucleotides, lacking the 3'-OH group required for forming a phosphodiester bond between two nucleotides during DNA chain extension. However, if each dideoxynucleotide carries different labels (e.g., 4 different fluorescent dyes), then all sequencing reactions can be carried out together without independent reaction.

[0041] Dideoxynucleotides are incorporated into nascent (i.e. extended) DNA chains to terminate DNA chain extension, producing nested DNA fragment groups of varying lengths. The newly synthesized and labeled DNA fragments are denatured and separated by size using gel electrophoresis on a denaturing polyacrylamide-urea gel that can resolve single-base differences in chain length. If each of the four DNA synthesis reactions is labeled with the same monochromatic marker (e.g., radioisotope), they are separated in one of four separate adjacent lanes in the gel, wherein each lane in the gel is named according to the dideoxynucleotides used in each reaction, i.e., gel lanes A, T, G, C. If four different markers are used, the reaction can be combined in a single lane on the gel. The DNA bands are then observed by autoradiography or fluorescence, and the DNA sequence can be read directly from X-ray film or gel image.

[0042] According to the identification of the terminal nucleotide bases added in the reaction, the band or its corresponding direct labeling is produced. The relative position of the different bands in the gel is then used to read the DNA sequence shown in (from the shortest to the longest). The Sanger sequencing process can be automated using a DNA sequencer, such as those commercially available from PerkinElmer, Beckman Coulter, Life Technologies, etc.

[0043] In other embodiments, the sequencing of nucleic acid is completed by single molecule sequencing using synthesis technology. Single molecule sequencing is for example in Lapidus et al. (U.S. Patent No. 7,169,560), Quake et al. (U.S. Patent No. 6,818,395), Harris (U.S. Patent No. 7,282,337), Quake et al. (U.S. Patent Application No. 2002 / 0164629) and Braslavsky et al., PNAS (USA), 100: 3960-3964 (2003) shows, and the content of each of these references is incorporated herein by reference as a whole. In short, single-stranded nucleic acid (such as DNA or cDNA) is hybridized with an oligonucleotide attached to the surface of a flow cell. Oligonucleotides can be covalently attached to the surface, or various attachments other than covalent attachment known to those of ordinary skill in the art can be adopted. In addition, attachment can be indirect, for example, by directly or indirectly attaching to a polymerase on the surface. The surface can be flat or other, and / or can be porous or non-porous, or any other type of surface suitable for attachment known to those of ordinary skill. The nucleic acids are then sequenced by imaging with single-molecule resolution the polymerase-mediated addition of fluorescently labeled nucleotides incorporated into oligonucleotides on the surface of the growing chain.

[0044] Other single molecule sequencing techniques involve detecting pyrophosphate as it is cleaved from a nascent strand of single nucleotide-incorporated DNA, as shown by Rothberg et al. (U.S. Pat. Nos. 7,335,762, 7,264,929, 7,244,559, and 7,211,390) and Leamon et al. (U.S. Pat. No. 7,323,305), the contents of each of which are incorporated herein by reference in their entirety.

[0045] In other embodiments, targeted resequencing is used. Resequencing is shown, for example, in Harris (U.S. Patent Application Nos. 2008 / 0233575, 2009 / 0075252, and 2009 / 0197257), the contents of each of which are incorporated herein by reference in their entirety. In short, a specific segment of the target is selected before sequencing (e.g., by PCR, microarray, or MIPS). Primers designed to hybridize to the specific segment are introduced and primer / template duplexes are formed. Under conditions sufficient for template-dependent nucleotide addition to the primers, the primer / template duplexes are exposed to a polymerase and at least one detectably labeled nucleotide. The incorporation of the labeled nucleotides and the identity of the nucleotides complementary to the nucleotides on the template at a position relative to the incorporated nucleotides are determined.

[0046] After the polymerization reaction, the primer can be removed from the duplex. The primer can be removed by any suitable method, such as by increasing the temperature of the surface or substrate to melt the duplex, or by changing the buffer conditions to make the duplex unstable, or a combination thereof. Methods for melting the template / primer duplex are well known in the art and are described in, for example, Chapter 10 of Molecular Cloning, a Laboratory Manual, 3rd Edition Supplement, J. Sambrook and DW Russell, Cold Spring Harbor Press (2001), the teachings of which are incorporated herein by reference.

[0047] After removing the primer, the template can be exposed to a second primer capable of hybridizing to the template. In one embodiment, the second primer is capable of hybridizing to the same region of the template (also referred to herein as the first region) as the first primer to form a template / primer duplex. The polymerization reaction is then repeated, thereby resequencing at least a portion of the template.

[0048] If the nucleic acid in the sample is degraded or only minimal nucleic acid can be obtained from the sample, PCR can be performed on the nucleic acid to obtain sufficient nucleic acid for sequencing (see, e.g., U.S. Pat. No. 4,683,195 to Mullis et al., the contents of which are incorporated herein by reference in their entirety).

[0049] Once the sequence of the rare nucleic acid is known, nucleic acid affinity reagents can be synthesized using conventional nucleic acid synthesis techniques in the art. Methods for synthesizing oligonucleotides are known in the art. See, for example, Sambrook et al. (DNA microarray: A Molecule Cloning Manual, Cold Spring Harbor, NY, 2003) or Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1982), the contents of which are incorporated herein by reference in their entirety. Suitable methods for synthesizing oligonucleotides are also described in Caruthers (Science 230: 281-285, 1985), the contents of which are incorporated herein by reference. Oligonucleotides can also be obtained from commercial sources, such as Operon Technologies, Amersham Pharmacia Biotech, Sigma and Life Technologies. Oligonucleotides can have the same melting temperature. The length of the oligonucleotide can be extended or shortened at the 5' end or the 3' end to produce an oligonucleotide with a desired melting temperature. Moreover, the annealing position of each oligonucleotide can be designed so that the sequence and length of the nucleic acid affinity reagent produce the desired melting temperature. The simplest equation for determining the melting temperature of a nucleic acid affinity reagent of less than 25 base pairs is the Wallace Rule (Td = 2(A + T) + 4(G + C)). Computer programs can also be used to design oligonucleotides, including but not limited to Array Designer software (Arrayit Inc.), oligonucleotide probe sequence design software for genetic analysis (Olympus Optical Co.), NetPrimer, and DNAsis from Hitachi Software Engineering. The TM (melting temperature) of each probe is calculated using a software program, such as Oligo Design available from Invitrogen Corp.

[0050] In other embodiments, affinity reagent can be an antibody. Antigens bound by antibodies can be determined from public documents. General methods for antibody production are described in Harlow et al. (Antibodies, Cold Spring Harbor Laboratory, pp. 93-117, 1988), including criteria to be considered when selecting animals for producing antiserum. For example, an animal of appropriate size is immunized by administering an immunogen that effectively produces an immune response, such as goats, dogs, sheep, mice or camels. An exemplary scheme is as follows. Depending on the size of the animal, 100 mg of antigen resuspended in an adjuvant (e.g., Freund's complete adjuvant) is injected into the animal, followed by a subcutaneous injection of 100 micrograms to 100 mg of immunogen and adjuvant (e.g., Freund's incomplete adjuvant) after three weeks, depending on the size of the animal. Additional subcutaneous or intraperitoneal injections are performed with adjuvants (e.g., Freund's incomplete adjuvant) every two weeks until a suitable antibody titer in the animal's blood is reached. Exemplary titers include a titer of at least about 1:5000 or a titer of 1:100,000 or higher, i.e., a dilution with detectable activity. For example, the antibody is purified by affinity purification on a column containing protein G resin or a target-specific affinity resin.

[0051] In vitro immunization techniques of human lymphocytes are used to produce monoclonal antibodies. Techniques for in vitro immunization of human lymphocytes are well known to those skilled in the art. See, for example, Inai et al., Histochemistry, 99(5):335-362, May 1993; Mulder et al., Hum. Immunol., 36(3):186-192, 1993; Harada et al., J. Oral Pathol. Med., 22(4):145-152, 1993; Stauber et al., J. Immunol. Methods, 161(2):157-168, 1993; and Venkateswaran et al., Hybridoma, 11(6)729-739, 1992. These techniques can be used to produce antigen-reactive monoclonal antibodies, including antigen-specific IgG and IgM monoclonal antibodies.

[0052] In certain embodiments, the conjugate moiety is an aptamer. As used herein, "aptamer" and "nucleic acid ligand" are used interchangeably and refer to a nucleic acid that has a specific binding affinity to a target molecule (e.g., a protein). Like all nucleic acids, a specific nucleic acid ligand can be described by a linear sequence of nucleotides (A, U, T, C, and G), typically 15-40 nucleotides long. Nucleic acid ligands can be engineered to encode a complementary sequence of a target protein known to be associated with the presence or absence of a specific disease.

[0053] In solution, the nucleotide chains form intramolecular interactions that fold the molecule into a complex three-dimensional shape. The shape of the nucleic acid ligand allows it to bind tightly to the surface of its target molecule. In addition to showing remarkable specificity, nucleic acid ligands generally bind their targets with very high affinities, for example, most anti-protein nucleic acid ligands have equilibrium dissociation constants in the picomolar to low nanomolar range.

[0054] The aptamer used in the composition of the present invention depends on the target tissue. Nucleic acid ligands can be found by any method known in the art. In one embodiment, nucleic acid ligands are found using an in vitro selection process called SELEX (systematic evolution of ligands by exponential enrichment). See, for example, Gold et al. (U.S. Patent Nos. 5,270,163 and 5,475,096), each of which is incorporated herein by reference in its entirety. SELEX is an iterative process for identifying nucleic acid ligands of selected molecular targets from a large number of nucleic acid libraries. The process relies on standard molecular biology techniques, using multiple rounds of selection, distribution and amplification of nucleic acid ligands to distinguish nucleic acid ligands with the highest affinity for target molecules. The SELEX method includes identifying high-affinity nucleic acid ligands containing modified nucleotides, which give ligands improved properties, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions of ribose and / or phosphate and / or base positions. Many improvements have been made to the basic SELEX method, any of which can be used to find nucleic acid ligands for the method of the present invention.

[0055] A "rare molecule" refers to a molecule that is present in a sample in a relatively small amount when compared to the amount of non-rare molecules in the sample. In some examples, the rare molecule accounts for about 10% of the total population of molecules in the sample suspected of containing the rare molecule. -8 wt % to about 10 -2 The amount of wt% is present. Rare molecules can be, but are not limited to, cell-free circulating molecules or molecules found in rare cells, such as malignant cells such as malignant tumors or cancer cells; circulating endothelial cells; circulating epithelial cells; mesenchymal cells; fetal cells; immune cells (B cells, T cells, macrophages, NK cells, monocytes); stem cells; nucleated red blood cells (normal erythroblasts or erythroblasts); and immature granulocytes. For example, rare molecules are further described in PCT / US2016 / 053610, the contents of which are incorporated herein by reference in their entirety.

[0056] Non-rare molecules are those molecules that are present in relatively large amounts when compared to the amount of rare molecules in a sample. In some examples, the non-rare molecules are at least about 10 times, or at least about 10 times, the amount of the rare molecule in the total population of molecules in a sample suspected of containing both non-rare molecules and rare molecules. 2 times, or at least about 10 3 times, or at least about 10 4times, or at least about 10 5 times, or at least about 10 6 times, or at least about 10 7 times, or at least about 10 8 The non-rare molecule can be a cell-free circulating non-rare molecule or a non-rare molecule found in non-rare cells, such as but not limited to white blood cells, platelets, and red blood cells.

[0057] For example, the term "rare molecule" may include, but is not limited to, antigens, proteins, peptides, hormones, vitamins, allergens, autoimmune antigens, carbohydrates, lipids, glycoproteins, cofactors, antibodies, enzymes, enzyme substrates, metabolites, nucleic acids, antibodies, organic amines, fatty acids, carbohydrates, cyclic hydrocarbons, aliphatic hydrocarbons, aromatic hydrocarbons, organic carboxylic acids, organic amines, neuroamines, cerebrosides, steroids, prostaglandins, carbohydrates, nucleosides and therapeutic drugs, as well as other biomolecules. For example, biomolecules for medical diagnosis of diseases are included, which include, but are not limited to, biomarkers for detecting cancer, heart damage, cardiovascular disease, neurological disease, hemostasis, fetal maternal assessment, fertility, bone status, hormone levels, vitamins, allergies, autoimmune diseases, hypertension, kidney disease, diabetes, liver disease, infectious diseases and other biomolecules that can be used for medical diagnosis of diseases. Biomarkers can be derived, for example, from cells such as bacteria, viruses, fungi, and protozoa; malignant cells, such as malignant tumors or cancer cells; circulating endothelial cells; circulating tumor cells; circulating cancer stem cells; circulating cancer mesenchymal cells; circulating epithelial cells; fetal cells; immune cells (B cells, T cells, macrophages, NK cells, monocytes); and stem cells.

[0058] For example, target rare molecules of rare cells include, but are not limited to, cancer cell type biomarkers, oncoproteins and oncogenes, chemoresistance biomarkers, metastatic potential biomarkers, and cell typing markers. For example, by way of illustration and not limitation, cancer cell type biomarkers include cytokeratins (CK) (CK1, CK2, CK3, CK4, CKS, CK6, CK7, CK8 and CK9, CK10, CK12, CK13, CK14, CK16, CK17, CK18, CK19, and CK2), epithelial cell adhesion molecules (EpCAM), N-cadherin, E-cadherin, and vimentin. For example, oncoproteins and oncogenes of potential therapeutic relevance due to mutations include, but are not limited to, WAF, BAX-1, PDGF, JAGGED 1, NOTCH, VEGF, VEGHR, CA1X, MIB1, MDM, PR, ER, SELS, SEMI, PI3K, AKT2, TWIST1, EML-4, DRAFF, C-MET, ABL1, EGFR, GNAS, MLH1, RET, MEK1, AKT1, ERBB2, HER2, HNF1A, MPL, SMAD4, ALK, ERBB4, HRAS, NOTCH 1. SMARCB1, APC, FBXW7, IDH1, NPM1, SMO, ATM, FGFR1, JAK2, NRAS, SRC, BRAF, FGFR2, JAK3, RA, STK11, CDH1, FGFR3, KDR, PIK3CA, TP53, CDKN2A, FLT3, KIT, PTEN, VHL, CSF1R, GNA11, KRAS, PTPN11, DDR2, CTNNB1, GNAQ, MET, RBI, AKT1, BRAF, DDR2, MEK1, NRAS, FGFR1, and ROS1.

[0059] For example, by way of illustration and not limitation, endothelial cell typing markers include CD136, CD105 / endoglin, CD144 / VE-cadherin, CD145, CD34, Cd41CD136, CD34, CD90, CD31 / PECAM-1, ESAM, VEGFR2 / Fik-1, Tie-2, CD202b / TEK, CD56 / NCAM, CD73 / VAP-2, Claudin 5, Z0-1, and Vimentin.

[0060] Metastatic potential biomarkers include, but are not limited to, urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), CD95, serine proteases (e.g., plasmin and ADAM); serine protease inhibitors (e.g., Bikunin); matrix metalloproteinases (e.g., MMP9); matrix metalloproteinase inhibitors (e.g., TIMP-1). By way of illustration and not limitation, chemoresistance biomarkers include PL2L piwi like, 5T4, ADLH, β-integrin, α6 integrin, c-kit, c-met, LIF-R, CXCR4, ESA, CD20, CD44, CD133, CKS, TRAF2, and ABC transporters, cancer cells lacking CD45 or CD31 but containing CD34 indicate cancer stem cells; and cancer cells containing CD44 but lacking CD24.

[0061] In the methods herein, leukocytes can be excluded as non-rare cells. For example, markers present on leukocytes (such as but not limited to CD45, CTLA-4, CD4, CD6S and CDS) can be used to indicate that the cell is not a rare cell of interest. In a specific non-limiting example, the CD45 antigen (also known as protein tyrosine phosphatase receptor type C or PTPRC) and originally called leukocyte common antigen can be used to detect all leukocytes.

[0062] In addition, CD45 can be used to distinguish different types of white blood cells that may be considered rare cells. For example, granulocytes are indicated by CD45+, CD15+; monocytes are indicated by CD45+, CD14+; T lymphocytes are indicated by CD45+, CD3+; T helper cells are indicated by CD45+, CD3+, CD4+; cytotoxic T cells are indicated by CD45+, CD3+, CDS+; β-lymphocytes are indicated by CD45+, CD19+ or CD45+, CD20+; thrombocytes are indicated by CD45+, CD61+; and natural killer cells are indicated by CD16+, CD56+ and CD3-. In addition, two commonly used CD molecules, CD4 and CD8, are usually used as markers for helper T cells and cytotoxic T cells, respectively. These molecules are defined in combination with CD3+ because some other white blood cells also express these CD molecules (some macrophages express low levels of CD4; dendritic cells express high levels of CDS).

[0063] In other cases, the rare cells are pathogens, including but not limited to Gram-positive bacteria (e.g., Enterococcus, Group B streptococcus, coagulase-negative staphylococci, Streptococcus viridans, Staphylococcus aureus, and saprophyticus, Lactobacillus and resistant strains thereof); yeast, including but not limited to, e.g., Candida albicans; Gram-negative bacteria, such as but not limited to, Escherichia coli, Klebsiella pneumoniae, Citrobacter koseri, Citrobacter freundii, Klebsiella oxytoca, Morganella morganii, Pseudomonas aeruginosa, Proteus mirabilis; mirabilis), Serratia marcescens, and Diphtheroids (gnb) and their resistant strains; viruses, such as but not limited to HIV, HPV, Flu, and MERSA; and sexually transmitted diseases. In the case of detecting rare cellular pathogens, a particle reagent is added that contains a binding partner that binds to a population of rare cellular pathogens. In addition, for each population of cellular target rare molecules on the pathogen, a reagent containing a binding partner for the cellular target rare molecule is added that binds to the cellular target rare molecule in the population.

[0064] The phrase "non-cell target rare molecule" refers to a target rare molecule that is not bound to a cell and / or circulates freely in a sample. For example, such non-cell target rare molecules include biomolecules used for medical diagnosis of diseases, including but not limited to biomarkers for detecting cancer, heart damage, cardiovascular disease, neurological disease, hemostasis, fetal maternal assessment, fertility, bone status, hormone levels, vitamins, allergies, autoimmune diseases, hypertension, kidney disease, diabetes, liver disease, infectious diseases, and other biomolecules that can be used for medical diagnosis of diseases.

[0065] An "MS marker precursor" is any molecule that produces an MS marker by the action of a modifying agent. An MS marker precursor may itself be an MS marker that is converted to another MS marker by the action of a modifying agent, such as by cleavage, by reaction with a moiety, by derivatization, or by addition or by subtraction of molecules, charges or atoms, or by a combination of two or more of the foregoing. An MS marker precursor includes a marker binding unit and a mass marker.

[0066] The "mass label" label includes a "charge unit" and a "mass unit".

[0067] A "charge unit" comprises a group of atoms arranged in such a way that one of them carries a formal charge. Figure 2 The structure shown in includes at least one charge unit X, which can be, for example, a NR moiety, such as a quaternary ammonium cation or at least one X is a quaternary phosphonium cation, where X=PR, where R can be hydrogen, alkyl or an aromatic organic derivative, such as methyl, benzyl or phenyl. Examples include carnitine, imidazolium, pyridinium, tetraethylamine, benzalkonium amine, alkylbenzeneethylammonium amine, triphenylphosphonium cation trialkyl (2,3-dihydroxypropyl) phosphonium salt or other quaternary structures.

[0068] The term "mass unit" comprises at least one atom as indicated by Y in Figure 3, such as CH, CH2, O, N, S, P, or a collection of atoms having a combined mass below 3 kDA, such as the phenyl group shown in 3a and 3b, and is attached to a "charge unit". For example, a variable mass unit may include structures of defined mass, such as polypeptides, polymers, carboxylic acids, carbohydrates, cyclic rings, aromatic rings, hydrocarbons, organic amines, nucleic acids, and organic alcohols, the mass of which may be altered by substitution and chain size. "Mass units" can include additional molecules, such as aromatic rings, peptides, fatty acids, carbohydrates, organic amines, nucleic acids, and organic alcohols (e.g., alkyl alcohols, acyl alcohols, phenols, polyols (e.g., diols), thiols, epoxides, primary, secondary, and tertiary amines, indoles, tertiary and quaternary ammonium compounds, amino alcohols, aminothiols, phenolic amines, indolecarboxylic acids, phenolic acids, vinylogous acids, carboxylates, phosphates, carboxylic acid amides, carboxylic acids from polyamides and polyesters, hydrazones, oximes, trimethylsilyl enol ethers, carbamates, ureas, guanidines, isocyanates, sulfonic acids, sulfonamides, sulfonylureas, sulfates, monoglycerides, glycerol ethers, sphingosine, which allow methods according to the principles described herein to be multiplexed to obtain more than one result at a time.

[0069] The "quaternary structure" and "mass structure" should be attached by bonds that are resistant to breaking, such as CC, CO, CN or other multivalent bonds. For example, Figure 4The carnitines shown in have carboxylic acids attached to peptide mass labels, such as L-carnitine-Phe-Ala-Gly-Gly-Ser-Cys, L-carnitine-Phe-Thr-Ala-Ser-Ala-Cys, L-carnitine-Phe-Phe-Ala-Ser-Cys, L-carnitine-Phe-Phe-Ala-Ser-Cys, L-carnitine-phe-gly-gly-ser-cys, L-carnitine-phe-phe-ser-gly-cys, L-carnitine-phe-gly-thr-thr-cys, L-carnitine-phe-gly-thr-ala-cys, carnitine-arg-gly-cys, and carnitine-phe-cys. In another example, quaternary mass-labeled methylimidazolium and pyridinium analogs are linked to aromatic mass labels via -CH2- bonds, such as Figure 5 shown.

[0070] The mass tag can be attached to the affinity reagent or rare molecule via a tag binding unit that promotes reversible bond formation, such as an acetal or ketal bond, as shown in Figure 3, panels AD. Figure 6 Shows how an affinity reagent or rare molecule represented by R is connected to a quadruple mass marker via an acetal or ketal bond. In this case, the marker binding unit is a diol produced by modification of an amine. The modification can be performed by reaction of an amine with β-hydroxy-γ-butyrolactone. Other reversible linking chemistries are known in the art and are suitable for the compositions of the present invention, such as those described in U.S. Patent Application Publication Nos. 2011 / 0294952 and 2016 / 0086781, the contents of each of which are incorporated herein by reference in their entirety.

[0071] These acetal or ketal bonds are destroyed by hydrolysis when exposed to aqueous acid, which is called a release agent in this case. For example, acetonitrile or water and an acid solvent will destroy the acetal or ketal bond and release the mass tag for analysis. It is important that the acetal or ketal bond will not break prematurely due to exposure to the liquid for affinity reaction, and is stable during storage. Usually, the combination between the labeling binding unit and the mass tag can be destroyed with any suitable release agent, which can be, for example, heating, ultrasonic treatment or a chemical reagent such as acid, depending on which one is suitable. The release agent also includes, but is not limited to, a chemical reagent, such as, but not limited to, acid, a catalyst (such as enzyme (including pseudoenzyme) and metal), an oxidant, a reducing agent, an acid, a base or other reagents that promote the rupture of the acetal or ketal bond.

[0072] One or more mass tags may be attached to a single affinity reagent, for example, for any single affinity reagent, any number from 1 to 3000 mass tags may be used. The advantage of having more than one mass tag associated with a single affinity unit is the ability to generate signal amplification. Primarily, this method amplifies the signal because many single molecules are generated to be measured by MS, and the method multiplies the signal by the number of single molecules generated. This may be important in rare event detection, where the target may only be present in less than 1% of the sample.

[0073] Quaternary mass tags produce unique mass fragments that, after ionization, fragment into at least one predictable mass that is analytically separated from the background. The mass tags and their product ions are detected inside the mass spectrometer. Figure 5 The respective fragmentation pathways for methylimidazolium and pyridinium are shown. Figure 7 The fragmentation pathways of the higher molecular weight derivatives are shown. The molecular weight of the quaternary mass marker was adjusted so that the unique mass fragments were not in the region of mass overlap with background components. Figure 5 Quaternary mass markers are shown with methylimidazolium (m / z 217) and pyridinium (m / z 214) as quaternary structures and an aromatic mass structure (m / z 135). The quaternary mass markers are due to fragmentation during collision-induced dissociation to produce the unique mass fragments shown, which are represented by methylimidazolium (m / z 83) and pyridinium (m / z 80). Figure 7 Quaternary mass markers with high molecular weight derivatives of imidazolium (m / z 309 and m / z 259) are shown. These quaternary mass markers produce higher mass fragments (m / z 175 and m / z 125) due to fragmentation upon ionization.

[0074] The predictability of the fragmentation of a precursor mass marker into one or more fragments (product ions) will depend on the chemical structure of the mass marker. For carbon-based mass markers, predictability follows certain rules. For example, the order of stability of carbocations is: primary < secondary < tertiary, with primary ions being less stable than secondary ions, which in turn are less stable than tertiary ions. Applying this logic to the fragmentation pattern means that splitting to produce secondary carbocations will be more successful than splitting to produce primary carbocations. Splitting to produce tertiary carbocations will still be more successful. Ions with a positive charge on the carbon of the carbonyl C=O are also relatively stable.

[0075] Typically, the following conventions will be used to analyze structures. Most fragments are even-electron cations. These cleavages produce more even-electron cations. The probability of cleaving a given bond is related to the bond strength and the stability of the fragments formed. Ten general rules to remember when predicting the most likely ions to form for a given molecule are as follows.

[0076] 1) For linear molecules, the relative height of the M+ peak is the largest and decreases with increasing branching.

[0077] 2) For the homologous series, the relative height of the M+ peak decreases with chain length.

[0078] 3) Cleavage is favored at the alkyl-substituted carbon, and the likelihood of cleavage increases with increasing substitution.

[0079] These rules are primarily derived from the fact that carbocation and radical stabilities exhibit the following trend: (most stable) benzyl > allyl > tertiary > secondary >> primary (least stable, "Stevenson's rule"). At the break point, the larger fragment generally leaves the radical behind the smaller cation.

[0080] 4) Double bonds, ring structures, and especially aromatic rings will stabilize molecular ions and increase the likelihood of their occurrence.

[0081] 5) The double bond favors allylic cleavage to produce a resonance-stabilized allylic carbocation, especially for cycloolefins.

[0082]

[0083] 6) For saturated rings (such as cyclohexane), the side chain tends to cleave first leaving the positive charge on the ring.

[0084]

[0085] 7) Unsaturated rings can also undergo retro-Diels-Alder reaction to eliminate neutral olefins.

[0086]

[0087] 8) Aromatic compounds tend to cleave to produce benzylic cations, or more likely quinonium cations.

[0088]

[0089] 9) C-C bonds immediately adjacent to heteroatoms often break, leaving a positive charge on the carbon bearing the heteroatom.

[0090]

[0091] 10) If small stable molecules such as water, CO, NH3, H2S, etc. can be driven out, then cracking is usually beneficial.

[0092] In addition to bond breaking, various intramolecular rearrangements can occur to produce sometimes unexpected ions. One common type of rearrangement in MS is the McLafferty rearrangement, which occurs in compounds containing a carbonyl group.

[0093]

[0094] General rules 1-3 apply very well to hydrocarbons. Rearrangements are common but do not usually produce strong peaks. The fragmentation pattern can be characterized by peaks separated by 14 mass units (CH2 groups). The most intense peaks are C3-C5 fragments. The MS of branched saturated hydrocarbons is similar, except that some fragments become more prominent. Cyclic hydrocarbons show more intense M+ ions (Rule 4) and two bonds must be broken to form fragments. It is usually easy to see the M+ peak of olefins. In non-cyclic olefins, double bonds migrate freely in the fragments, so it may be difficult to determine the double bond position, but it is easier for cyclic olefins. Fragmentation usually occurs at allylic bonds (Rule 5). Aromatic hydrocarbons usually show strong M+ peaks (Rule 4). Aromatic rings are stable and have a low tendency to fragment. Alkyl substituted benzenes usually produce a strong peak at m / z 91 due to benzylic cleavage (Rule 8).

[0095] Alcohols fragment very easily - secondary and primary alcohols show very weak M+ peaks, while tertiary alcohols usually show no M+ at all. MW is usually determined by derivatization. The CC bond closest to the OH is usually the first bond to break. Therefore, primary alcohols usually show a prominent peak at 31 m / z. Secondary alcohols fragment in the same way, usually showing a prominent + CHR-OH peak. Sometimes, the hydrogen R2CH-OH in the 1° and 2° alcohols cleaves instead of the alkyl group. The result is the M-1 peak. Tertiary alcohols cleave in a similar manner to give + CRR-OH fragments. Alcohols may lose a molecule of water to show a sometimes prominent M-18 peak. This is especially evident for primary alcohols. Benzyl alcohol fragments in a very different manner than aliphatic alcohols. Benzyl cleavage occurs as expected (Rule 8). Benzyl alcohol typically loses water (M-18). The M-18 peak is particularly strong for molecules where the loss of water is mechanistically simple. Phenol typically shows a peak at 77 m / z resulting from the formation of the phenyl cation, and peaks resulting from the loss of CO (M-28) and CHO (M-29) are often present in phenol. If other attractive fragmentation pathways are available.

[0096] For ethers, cleavage occurs in two main ways: C-C bond cleavage next to O (such as alcohols); or C-O bond cleavage, and the charge is on the C fragment. For aromatic ethers, M+ is usually strong. MS is similar to phenol - both form a phenoxy cation (m / z 93) and related daughters.

[0097] Ketones usually give rise to a strong M+ peak. The main fragmentation pathway involves α-cleavage to produce an acyl ion. Fragments containing a carbonyl group may also carry a free radical. For ketones with longer chains, the McLaren rearrangement usually results in a strong peak. For aromatic ketones, M+ is prominent. The primary cleavage is α to the carbonyl group to produce a strong ArCO+ peak (m / z 105 when Ar=Ph). This will lose CO to give a phenyl cation (m / z 77).

[0098] Aldehydes show a weak but discernible M+ peak. The major pathways are α-cleavage and McLaurel rearrangement. Aromatic aldehydes are similar to aromatic ketones. M+ is strong, and M-1 (α-cleavage to carbonyl) is also strong, giving the ArCO+ ion (Ar=Ph, m / z 105). Loss of CO from this ion is common to give the m / z 77 phenyl cation.

[0099] For carboxylic acids, the M+ is weak and not always visible. A characteristic m / z 60 peak is often present due to the McLaren rearrangement. The bond alpha to the carbonyl is also often broken, giving M-OH and M-CO2H peaks.

[0100] For aromatic acids, M+ is very prominent. Common peaks are the loss of OH (M-17) and the loss of CO2H (M-45). If ortho hydrogen groups are present, then the loss of water (M-18) may also be seen.

[0101] For aliphatic esters, M+ is usually different. The most distinctive peak is due to the McLaren rearrangement. Unless the RO chain is very long, M+ is usually prominent. The base peak is the loss of RO. The McLaren rearrangement produces the corresponding acid. More complex rearrangements usually produce a prominent acid +1 peak.

[0102] Aliphatic monoamines have weak odd-numbered M+ peaks. The most important fragmentation is usually the breakage of a CC bond next to a CN bond. The base peak of almost all primary amines occurs at 30 m / z. For aromatic amines, M+ is intense. The NH bond can be broken to produce a moderately intense M-1 peak. Common fragmentations are the loss of HCN and H2CN, producing peaks at 65 and 66 m / z. Alkyl-substituted aromatic amines usually show breakage of a CC bond next to CN, producing a strong peak at 106 when Ar=Ph.

[0103] Primary amides give a strong peak at m / z 44 due to the cleavage of the R-CONH2 bond. Aliphatic amides -M+ are weak but discernible. For linear amides with more than 3 carbons, the McLaren Spectrophotometer gives a base peak at m / z 59.

[0104] For nitriles, the M+ of aliphatic nitriles is weak or absent. Loss of the α-hydrogen can produce a weak M-1 peak. Due to rearrangements like McLaren's, the base peak is usually at 41. This has limited diagnostic value since (C3H5+) has the same mass.

[0105] Aliphatic nitro compounds have a weak odd M+. The main peaks are hydrocarbon fragments up to M-NO2. For aromatic nitro compounds, M+ is very strong. The prominent peaks result from the loss of the NO2 radical, resulting in the M-46 peak. Also prominent is M-30, which is the loss of NO.

[0106] Fig. 9 Describes a method for predicting the fragmentation of peptide mass markers in a mass spectrometer. The fragmentation of a peptide (amino acid chain) usually occurs along the peptide backbone. Each residue of the peptide chain separates successively in the N->C and C->N directions. The side chains of the amino acids are different. Fig. 9 As shown, based on different side chains, each amino acid has the weakest bond, which leads to the predictable breakage of the peptide. Peptides have a tendency to break at Asp (D) (Mass Spectrometry in Proteomics Ruedi Aebersold* and David R. Goodlett 269 Chem. Rev. 2001, 101, 269-295). Predicting the breakage of peptides in mass spectrometers is further described in, for example, Beardsley et al. (Journal of the American Society for Mass Spectrometry, 15 (2): 158-167, 2004), the contents of which are incorporated herein by reference in their entirety. Peptide breakage algorithms are also commercially available. Exemplary algorithms for predicting peptide breakage are described by Zhang (Analytical Chemistry 76, 3908-3922, 2004) or Zhang (Analytical Chemistry 77, 6364-6373, 2005), the contents of which are incorporated herein by reference in their entirety.

[0107] In certain embodiments, the peptide is labeled at the N-terminus, C-terminus, or any internal amino acid residue. For example, a commercially available small cysteine-terminated peptide (e.g., YGMTSR*YFC, in which arginine is fully labeled with 13C6 and 14N4) can be selected as a mass marker.

[0108] The term "altering agent" or "releasing agent" refers to a substance that can change the MS marker precursor, destroy the bond of the marker binding unit to release the mass marker. In certain embodiments, the altering agent can interact with the MS marker precursor to obtain an MS marker with a unique mass in the range of about 1Da to about 3kDa, or in the range of about 1Da to about 50Da, or in the range of about 50Da to about 150Da, or in the range of about 150Da to about 700Da, or in the range of about 700Da to about 3kDa. In some instances, the unique mass of the MS marker is less than about 3kDa. The MS marker precursor can be changed by bond destruction to form neutral, negative or positive ions or free radicals. Changing the MS marker precursor by an altering agent can be by adding atoms, charges or electrons to the MS marker precursor, or subtracting atoms, charges or electrons from the MS marker precursor, or by bond cleavage or bond formation in the MS marker precursor. Altering agents include, but are not limited to, chemical reagents, such as, but not limited to, catalysts (such as enzymes (including pseudoenzymes) and metals), oxidants, reducing agents, acids, bases, agents that promote substitution reactions or replacement reactions; and ionizing agents. In some examples, the altering agent promotes the formation of an MS tag from an MS tag precursor, such as by promoting reaction of the MS tag precursor with a moiety to form an MS tag. In some examples, the altering agent promotes the formation of an MS tag from an MS tag precursor, such as by promoting release of the MS tag from the MS tag precursor.

[0109] The properties of the MS marker precursor may depend on, for example, one or more of the following: the properties of the MS marker, the properties of the MS method used, the properties of the MS detector used, the properties of the target rare molecule, the properties of the affinity agent, the properties of any immunoassay used, the properties of the sample, the properties of any buffer used, the properties of the separation. In some instances, the MS marker precursor is a molecule whose mass can be changed by substitution and / or chain size. The MS marker produced by the MS marker precursor is a molecule of limited mass, which should not be present in the sample to be analyzed. In addition, the MS marker should be within the range of detection of the MS detector, should not have overlapping masses and should be detectable by primary mass. By way of illustration and not limitation, examples of MS labeling precursors for use in the methods of the invention include, by way of illustration and not limitation, polypeptides, organic and inorganic polymers, fatty acids, carbohydrates, cyclic hydrocarbons, aliphatic hydrocarbons, aromatic hydrocarbons, organic carboxylic acids, organic amines, nucleic acids, organic alcohols (e.g., alkyl alcohols, acyl alcohols, phenols, polyols (e.g., diols), thiols, epoxides, primary, secondary and tertiary amines, indoles, tertiary and quaternary ammonium compounds, amino alcohols, aminothiols, phenolamines, indolecarboxylic acids, phenolic acids, vinylogous acids, carboxylates, phosphates, carboxylic acid amides, carboxylic acids from polyamides and polyesters, hydrazones, oximes, trimethylsilyl enol ethers, acetals, ketals, carbamates, ureas, guanidines, isocyanates, sulfonic acids, sulfonamides, sulfonylureas, sulfates, monoglycerides, glycerol ethers, sphingoid bases, neuramines, cerebrosides, steroids, prostaglandins, carbohydrates, nucleosides, and therapeutic drugs.

[0110] The MS marker precursor may include 1 to about 100,000 MS markers, or about 10 to about 100,000 MS markers, or about 100 to about 100,000 MS markers, or about 1,000 to about 100,000 MS markers, or about 10,000 to about 100,000 MS markers. The MS marker precursor may be composed of a protein, polypeptide, polymer, particle, carbohydrate, nucleic acid, lipid or other macromolecule capable of including multiple MS marker repeating units by attachment. Multiple MS markers allow amplification because each MS marker precursor can produce many MS markers.

[0111] For example, for polypeptide MS labeling precursors, the chain length of the polypeptide can be adjusted to produce MS labels in the mass region without background peaks. In addition, MS labels can be produced by MS labeling precursors with unique mass, which are not present in the test sample. Polypeptide MS labeling precursors can include additional amino acids or derived amino acids, which allow method multiplexing to obtain more than one result at a time. Examples of polypeptide MS labeling precursors include, but are not limited to, for example, polyglycine, polyalanine, polyserine, polythreonine, polycysteine, polyvaline, polyleucine, polyisoleucine, polymethionine, polyproline, polyphenylalanine, polytyrosine, polytryptophan, polyaspartic acid, polyglutamic acid, polyasparagine, polyglutamine, polyhistidine, polylysine and polyarginine. Polypeptide MS labeling precursors distinguished by a mixture of amino acids or derived amino acids produce masses with or without free radicals with even or odd selected ions. In some instances, polypeptides can be modified by catalysis. For example, as an illustration and not limitation, peroxidase can be used as a catalyst to add phenol and aromatic amines to polythreonine. In another example, by way of illustration and not limitation, a peroxidase can be used as a catalyst to transfer electrons to an aromatic amine. In another example, by way of illustration and not limitation, a phosphatase can be used as a catalyst to remove a phosphate from an organophosphate.

[0112] In another example, by way of illustration and not limitation, a derivatizing agent is used as part to generate an MS tag from an MS tag precursor. For example, dinitrophenyl and other nitrophenyl derivatives can be formed from an MS tag precursor. By way of illustration and not limitation, other examples include esterification, acylation, silylation, protective alkylation, derivatization by keto-base condensation such as Schiff base, cyclization, formation of fluorescent derivatives and inorganic anions. The derivatization reaction can occur before MS analysis, but in a microreaction after the affinity reaction or be used to generate an MS tag precursor conjugated to an affinity reagent.

[0113] In some examples, the MS labeling precursor may include isotopes such as, but not limited to 2 H.13 C and 18 O, which is retained in the MS tag derived from the MS tag precursor. The MS tag can be detected by the primary mass or the secondary mass after ionization. In some examples, the MS tag precursor is a precursor with a relatively high probability of initiating bond cleavage, such as but not limited to alkylated amines, acetals, primary amines and amides, for example, where the MS tag can produce masses with or without free radicals with even or odd selected ions. The selection of polypeptides can produce unique MS spectral features.

[0114] As mentioned above, the change agent can be an enzyme (including pseudoenzyme). In some instances, catalysis can be carried out under the water-insoluble enzyme derivative fixed with for example the following things: silica gel, charcoal, DEAE-cellulose, DEAE-SEPHADEX (cross-linked dextran gel, available from Sigma Aldrich), cellulose citrate, kaolin, cellulose phosphate, acid clay, AMBERLITE XE-97 (carboxylic acid cation exchange resin manufactured by Rohm & Haas), carboxymethyl cellulose, glass, quartz, dowex-50, starch gel, polyacrylamide gel, polyamino acid or aminobenzyl cellulose. Cross-linking agent can be used for fixing enzyme. For example, this cross-linking agent includes but is not limited to glutaraldehyde, dimethyl adipimidate, carbodiimide, maleic anhydride, MDA methylene dianiline, hydrazide and acyl azide.

[0115] In some examples, an enzyme used for the purpose consistent with the principles described herein is any enzyme with a high turnover rate that can convert an enzyme substrate (e.g., a MS marker precursor) into an MS marker that is detected by the mass detector of a mass spectrometer in the presence of unconverted substrate. The enzyme should not be in the sample being tested, or if present in the sample, must be removed from the sample prior to testing. Examples of enzymes suitable for this purpose include, but are not limited to, phosphatases (e.g., alkaline phosphatases, lipid phosphatases, tyrosine phosphatases, serine phosphatases, threonine phosphatases, and histidine phosphatases); oxidases (e.g., horseradish peroxidase, copper amine oxidase, D-amino acid oxidase, galactose oxidase, plasma amine oxidase, tryptophan peroxidase, uricase oxidase, and xanthine oxidase); β-galactosidase; transferases (e.g., D-alanine transferases, glycosyltransferases, acyltransferases, alkyltransferases, aryltransferases, one-carbon transferases, ketonetransferases, aldehydetransferases, nitrogenous transferases, phosphorustransferases, sulfurtransferases, and pentosyltransferases); peptidases (e.g., pepsin, papain, chymosin (rennin), renin, thrombin, trypsin, matrix metallopeptidase, cathepsin, cysteine acid proteases and carboxypeptidases); aldolases (e.g., carboxy aldolases, aldolases, oxoacids, tryptophanases); fatty acid enzymes (e.g., fatty acid amine hydrolases, fatty acid synthases, and choline acetyltransferases), and combinations of two or more thereof (e.g., alkaline phosphatases, acid phosphatases, oxidases, β-galactosidases, peroxidases, acylases, asparaginases, catalases, chymotrypsin, amylases, glucoamylases, glucose oxidases, glucose-6-phosphate dehydrogenases, hexokinases, invertases, lipases, phosphoglucomutases, ribonucleases, acetylcholinesterases, alcohol dehydrogenases, aldolases, cholinesterases, citrate synthases, ureases, amyl glucosidases, carboxypeptidases, cholinesterases, luciferases, ribonucleases, pyruvate kinases, and subtilisin).

[0116] The substrate for the enzyme is an MS label precursor, which contains an MS label released by the action of the enzyme on the substrate. As an illustration and not limitation, such MS labels that can be part of the enzyme substrate include, for example, phenols (from substrates such as p-nitrophenyl phosphate, p-nitrophenyl-β-D-galactoside, amino acids, peptides, carbohydrates (6-phospho-D-gluconate), fatty acids (acetyl-CoA), alkylamines, glycerol); and naphthols (from substrates such as p-nitronaphthyl phosphate, p-nitro-naphthyl-β-D-galactoside).

[0117] Metals that can be used to release the MS label from the portion attached to the affinity agent include, but are not limited to, for example, transition metals (e.g., palladium, platinum, gold, ruthenium, rhodium or iridium), chelated metals (e.g., iron, copper, cobalt, magnesium complexed with ethylenediaminetetraacetate (EDTA), N-(2-hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA) or trans-1,2-cyclohexanediaminetetraacetic acid (CDTA)), metal oxidants (e.g., sodium hypochlorite, potassium periodate, silver oxide, chromic acid, potassium permanganate and sodium perborate) and metal reducing agents (e.g., lithium aluminum hydride, sodium borohydride, sodium ascorbate, phosphites and sodium).

[0118] MS mark can be directly detected, or the released MS mark can further react with another compound to form a derivative MS mark, which is detected by MS technology. The derivative MS mark is a compound formed by the MS mark obtained from the MS mark precursor, wherein the compound can also be detected by MS technology. This method of forming a derivative MS mark further enhances the multiplexing capability of the method according to the principles described herein. For example, the released phenol or naphthol can be coupled with aromatic amines in the presence of peroxidase (see, for example, U.S. Patent number 5,182,213, the relevant disclosure of which is incorporated herein by reference). In an example, the released naphthol is coupled with phenylenediamine (such as α-phenylenediamine dihydrochloride) in an alkaline medium in the presence of a peroxidase active substance to produce a derivative MS mark. Multiplexing can be achieved using different naphthols and / or different phenylenediamines.

[0119] Internal standards are an important aspect of mass spectrometry. In some instances, in addition to the MS label used to detect rare target molecules, a second mass label (as an internal standard) can be added that can be measured. The internal standard has a similar structure to the MS label, with a slightly altered mass. Internal standards can be prepared that contain additional amino acids or derivatized amino acids. Alternatively, internal standards can be prepared by incorporating isotopic labels, such as, but not limited to, 2 H(D), 13 C and 18 O. The mass of the MS isotope label is higher than the naturally occurring substance. For example, isotope labeled MS labels such as glycerol-C-d7, sodium acetate-C-d7, sodium pyruvate-C-d7, D-glucose-C-d7, deuterated glucose, and dextrose-C-d7 will serve as internal standards for glycerol, sodium acetate, sodium pyruvate, glucose, and dextrose, respectively.

[0120] The MS labeling precursor or the change agent can be covalently attached to the affinity agent directly through a bond or through an intermediate linking group (to produce a modified affinity agent). In some embodiments, the preparation of the modified affinity agent can be carried out by using a functional group suitable for attaching the MS labeling precursor or the change agent to the affinity agent through a direct bond. The nature of the functional group used depends on, for example, one or more of the following: the nature of the MS labeling precursor, the nature of the change agent, and the nature of the affinity agent, including, for example, the nature of one or more different particles of the carrier particles and the labeling particles that can be part of the affinity agent. A large number of suitable functional groups can be used for attachment to amino groups and alcohols; such functional groups include, for example, activated esters, including, for example, carboxylates, imine esters, sulfonates, and phosphates; activated nitrites; aldehydes; ketones; and alkylating agents.

[0121] The linking group can be a chain having 1 to about 60 or more atoms or 1 to about 50 atoms or 1 to about 40 atoms or 1 to 30 atoms or about 1 to about 20 atoms or about 1 to about 10 atoms, and the atoms are each independently selected from the group usually composed of carbon, oxygen, sulfur, nitrogen and phosphorus, usually carbon and oxygen. The number of heteroatoms in the linking group can be about 0 to about 8, about 1 to about 6, or about 2 to about 4. The atoms of the linking group can be replaced by atoms other than hydrogen, such as one or more of carbon, oxygen and nitrogen in the form of alkyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy or aralkyloxy. As a general rule, the length of a specific linking group can be arbitrarily selected to provide the convenience of synthesis, provided that the interference caused by the linking group is minimal under the function related to the method disclosed herein that the molecule connected can perform.

[0122] The linking group may be aliphatic or aromatic. When heteroatoms are present, oxygen will generally be present as an oxy or oxo group, bonded to carbon, sulfur, nitrogen or phosphorus; sulfur will generally be present as a thioether or thiocarbonyl group; nitrogen will generally be present as a nitro, nitroso or amino group, generally bonded to carbon, oxygen, sulfur or phosphorus; phosphorus will generally be bonded to carbon, sulfur, oxygen or nitrogen, generally in the form of phosphonates and phosphate monoesters or diesters. The functional groups present in the linking group may include esters, thioesters, amides, sulfamides, ethers, ureas, thioureas, guanidines, azo groups, thioethers, carboxylates and the like. The linking group may also be a macromolecule, such as a polysaccharide, a peptide, a protein, a nucleotide and a dendrimer.

[0123] In certain embodiments, MS labeling precursor or change agent (as the case may be) and affinity agent can be non-covalently linked together. Adopt binding pair, usually specific binding pair member, one of which is connected to affinity agent and another member is connected to MS labeling precursor or change agent. Binding pair member's combination causes affinity agent and MS labeling precursor or change agent non-covalent connection. Binding pair member can be directly connected to one or both of MS labeling precursor or change agent and affinity agent, or be indirectly connected by intermediate linking group, and the property of linking group is discussed above. In some instances, the member of specific binding pair has relatively high binding constant, for example, as an illustration and not limitation, avidin (streptavidin)-biotin binding, fluorescein (FITC) and the antibody for FITC, rhodamine (Texas Red) and the antibody for rhodamine, digitonin (DIG) and the antibody for DIG, non-human species antibody (for example goat, rabbit, mouse, chicken, sheep) and anti-species antibody.

[0124] The modified affinity agents can be prepared by linking each different affinity agent to an MS labeling precursor or altering agent in a separate individual reaction and then combining the modified affinity agents to form a mixture comprising the modified affinity agents. Alternatively, different affinity agents can be combined and the reaction linking the affinity agents to the MS labeling precursor or altering agent can be performed when combined. This allows the method to be multiplexed to obtain more than one result at a time.

[0125] The amount of each different modified affinity agent used in the methods of the present invention depends on, for example, one or more of the following: the nature and possible amount of each different target rare molecule population, the nature of the MS label, the nature of the affinity agent, the nature of the cells (if present), the nature of the particles (if used), and the amount and nature of the blocking agent (if used). In some examples, the amount of each different modified affinity agent used is about 0.001 μg / μL to about 100 μg / μL, or about 0.001 μg / μL to about 80 μg / μL, or about 0.001 μg / μL to about 60 μg / μL, or about 0.001 μg / μL to about 40 μg / μL, or about 0.001 μg / μL to about 20 μg / μL, or about 0.001 μg / μL to about 15 μg / μL. 001 μg / μL to about 10 μg / μL, or about 0.5 μg / μL to about 100 μg / μL, or about 0.5 μg / μL to about 80 μg / μL, or about 0.5 μg / μL to about 60 μg / μL, or about 0.5 μg / μL to about 40 μg / μL, or about 0.5 μg / μL to about 20 μg / μL or about 0.5 μg / μL to about 10 μg / μL.

[0126] The quantity of the change agent adopted can be one for each MS label precursor, or one for every two MS label precursors, or one for every three MS label precursors, at most one for all the MS label precursors adopted, depending on the property of the MS label precursor, the property of the change agent, whether the change agent is free or a part of the modified affinity agent in the medium, and one or more of the properties and quantities of the different affinity agents used. For example, when each MS label precursor comprises an unstable ester bond or unstable amide bond of different MS labels and affinity agents, a single change agent causing disulfide bond, ester bond or amide bond hydrolysis to obtain different MS labels can be adopted. In other examples, a kind of change agent can be utilized, or a change agent less than the quantity of the MS label precursor can be adopted. In another example, different change agents can be used to produce MS labels for each different type of affinity agents used.

[0127] Combination comprising sample (optionally concentrated) and modified affinity agent in aqueous medium is handled by being maintained at the temperature that makes modified affinity agent and target rare molecule combination on cell or particle reagent for a period of time.For every kind of modified affinity agent comprising changer, the MS labeling precursor that changer acts on is included in the combination, and wherein MS labeling precursor is converted into MS labeling.In some instances, add other part, wherein changer promotes the reaction of part and MS labeling precursor to produce MS labeling.In some instances, modified affinity agent comprises MS labeling precursor, and changer is included in the combination as the unbound material in medium.In this example, changer acts on the MS labeling precursor of affinity agent to produce MS labeling.In some instances, adopt the first changer of the entity that releases the MS labeling precursor from affinity agent, and adopt the second changer to promote to form MS labeling from MS labeling precursor subsequently.

[0128] The temperature and duration of this treatment depend on the properties of, for example, the properties of the sample, the properties of the target rare molecules, the properties of the non-rare molecules, the properties of the modified affinity agents, the properties of the MS labeling precursors and the properties of the change agents. In some instances, medium temperatures and usually constant temperatures are generally adopted, preferably room temperature. The temperature during the maintenance period is generally, for example, about 5°C to about 99°C or about 15°C to about 70°C, or about 20°C to about 45°C. The holding time is, for example, about 0.2 seconds to about 24 hours, or about 1 second to about 6 hours, or about 2 seconds to about 1 hour, or about 1 to about 15 minutes. The time period depends on, for example, the temperature of the medium and the binding rate of various reagents.

[0129] Modified affinity agents that have bound to the target rare molecule, ie, affinity agents for which the modifying agent has acted, are optionally separated from modified affinity agents that have not bound to the target molecule. In some examples, this separation involves reducing the number of non-rare molecules in the sample.

[0130] sample

[0131] The compositions of the present invention can be used to analyze many different types of samples. A variety of heterogeneous samples can be analyzed, such as biological samples, environmental samples (including, for example, industrial samples and agricultural samples), and food / beverage product samples, etc.

[0132] Exemplary environmental samples include, but are not limited to, groundwater, surface water, saturated soil water, unsaturated soil water; industrial processes, such as wastewater, cooling water; chemicals used in processes, chemical reactions in industrial processes, and other systems involving leachate from waste sites; wastewater and water injection processes; leak detection of liquids in or around storage tanks; discharged water from industrial facilities, water treatment plants or facilities; drainage and leachate from agricultural fields, drainage from urban land uses, such as surface, underground and sewer systems; water from waste treatment technologies; and drainage from mineral mining or other processes for extracting natural resources such as oil production and in situ energy production.

[0133] In addition, exemplary environmental samples include, but are not limited to, agricultural samples, such as crop samples, such as grains and feed products, such as soybeans, wheat, and corn. Typically, data on product composition is required, such as moisture, protein, oil, starch, amino acids, extractable starch, density, test weight, digestibility, cell wall content, and any other components or characteristics of commercial value.

[0134] Exemplary biological samples include human tissue or body fluid, and can be collected in any clinically acceptable manner. Tissue is a large number of connected cells and / or extracellular matrix materials, such as derived from skin tissue, hair, nail, nasal tissue, CNS tissue, neural tissue, eye tissue, liver tissue, kidney tissue, placental tissue, breast tissue, placental tissue, breast tissue, gastrointestinal tissue, musculoskeletal tissue, urogenital tissue, bone marrow, etc., such as humans or other mammals, including connecting materials and liquid materials related to cells and / or tissues. Body fluid is a liquid material derived from, for example, humans or other mammals. These body fluids include but are not limited to mucus, blood, blood plasma, serum, serum derivatives, bile, blood, maternal blood, sputum, saliva, sputum, sweat, amniotic fluid, menstrual fluid, milk, peritoneal fluid, urine, semen and cerebrospinal fluid (CSF), such as lumbar vertebrae or ventricular CSF. Sample can also be fine needle aspiration or biopsy tissue. Sample can also be a culture medium containing cells or biomaterials. Sample can also be a blood clot, such as a blood clot obtained from whole blood after removing serum.

[0135] In one embodiment, the biological sample can be a blood sample, from which plasma or serum can be extracted. Blood can be obtained by standard phlebotomy and then separated. Typical separation methods for preparing plasma samples include centrifuging blood samples. For example, protease inhibitors and / or anticoagulants are added to the blood sample immediately after blood is drawn. The tube is then cooled and centrifuged and can then be placed on ice. The resulting sample is separated into the following components: a clear solution of plasma in the upper phase; a buffy coat, which is a thin layer of white blood cells mixed with platelets; and red blood cells. Typically, 8.5 mL of whole blood will produce about 2.5-3.0 mL of plasma.

[0136] Serum is prepared in a very similar manner. Venous blood is collected, and then protease inhibitors and coagulants are mixed with the blood by inversion. The blood is allowed to clot by standing the tube vertically at room temperature. The blood is then centrifuged, with the resulting supernatant being designated serum. The serum sample should then be placed on ice.

[0137] Before analyzing the sample, the sample can be purified, for example, using filtration or centrifugation. For example, these techniques can be used to remove particles and chemical interferences. Various filter media for removing particles include filter paper, such as cellulose and membrane filters, such as regenerated cellulose, cellulose acetate, nylon, PTFE, polypropylene, polyester, polyethersulfone, polycarbonate and polyvinylpyrrolidone. Various filter media for removing particles and matrix interference include functionalized membranes, such as ion exchange membranes and affinity membranes; SPE filter cartridges, such as silica gel and polymer-based filter cartridges; and SPE (solid phase extraction) discs, such as PTFE and glass fiber-based. Some of these filters can be provided in disc form for loose placement in a filter holder / housing, other filters are provided in disposable tips that can be placed on, for example, standard blood collection tubes, and some are provided in arrays with holes for receiving pipetted samples. Another type of filter includes a spin filter. The spin filter consists of a polypropylene centrifuge tube with a cellulose acetate filter membrane and is used in conjunction with centrifugation to remove particles from a sample (e.g., serum and plasma samples that are typically diluted in an aqueous buffer).

[0138] Filtration is partially affected by the porosity value, such that larger porosities only filter out larger particles and smaller porosities filter out both smaller and larger ones. Typical porosity values ​​for sample filtration are 0.20 and 0.45 μm porosity. Samples containing colloidal materials or large amounts of fine particles may require considerable pressure to force the liquid sample through the filter. Therefore, for samples such as soil extracts or wastewater, a prefilter or depth filter bed (such as a "2-in-1" filter) may be used and placed on top of the membrane to prevent clogging by samples containing these types of particles.

[0139] In some cases, centrifugation without a filter can be used to remove particles, which is often done with urine samples. For example, the sample is centrifuged. The resulting supernatant is then removed and frozen.

[0140] After obtaining and purifying the sample, the sample can be analyzed with the composition of the present invention to detect one or more target analytes, such as rare molecules in a plasma sample. Regarding the analysis of plasma samples, there are many elements in the plasma, such as proteins (e.g., albumin), ions and metals (e.g., iron), vitamins, hormones, and other elements (e.g., bilirubin and uric acid). Any of these elements can be detected using the composition of the present invention. More specifically, the composition of the present invention can be used to detect molecules indicating disease states in biological samples. Specific examples are provided below.

[0141] When one or more target rare molecules are part of a cell, the aqueous medium can also include a lysing agent for lysing cells. A lysing agent is a compound or a mixture of compounds that destroys the integrity of the cell membrane to release the intracellular contents of the cell. Examples of lysing agents include, but are not limited to, for example, nonionic detergents, anionic detergents, amphoteric detergents, low ionic strength aqueous solutions (hypotonic solutions), bacterial agents, fatty aldehydes, and antibodies that cause complement dependent lysis. Various auxiliary materials may be present in the dilution medium. All materials in the aqueous medium exist in a concentration or amount sufficient to obtain a desired effect or function.

[0142] In some instances, when one or more target rare molecules are part of a cell, it may be necessary to fix the cells of the sample. Fixation of cells fixes the cells and maintains the cell structure and maintains the cells under conditions similar to those of cells in vivo and under conditions under which the antigen of interest can be recognized by a specific affinity agent. The amount of the fixative used is an amount that keeps the cells but does not cause erroneous results in subsequent determinations. The amount of the fixative may depend on, for example, one or more of the properties of the fixative and the properties of the cells. In some instances, the amount of the fixative is about 0.05% to about 0.15% or about 0.05% to about 0.10% or about 0.10% to about 0.15% by weight. Reagents for fixing cells include, but are not limited to, for example, crosslinking agents, such as aldehyde reagents (such as, for example, formaldehyde, glutaraldehyde, and paraformaldehyde); alcohols (e.g., C1-C5 alcohols, such as methanol, ethanol, and isopropanol); ketones (e.g., C3-C5 ketones, such as acetone). The name C1-C5 or C3-C5 refers to the number of carbon atoms in the alcohol or ketone. The fixed cells may be subjected to one or more washing steps using a buffered aqueous medium.

[0143] If desired, after fixation, the cell preparation can also be permeabilized. In some cases, fixatives such as alcohols (e.g., methanol or ethanol) or ketones (e.g., acetone) can also cause permeabilization, and no additional permeabilization step is required. Permeabilization provides access to the target molecule of interest through the cell membrane. The amount of the permeabilizing agent used is the amount that destroys the cell membrane and allows entry of the target molecule. The amount of the permeabilizing agent depends on one or more of the properties and amount of the permeabilizing agent and the cell. In some instances, the amount of the permeabilizing agent is from about 0.01% to about 10%, or from about 0.1% to about 10%. Reagents for cell permeabilization include, but are not limited to, alcohols (e.g., C1-C5 alcohols, such as methanol and ethanol); ketones (e.g., C3-C5 ketones, such as acetone); detergents (e.g., saponin, TRITON X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, tert-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether buffer, available from Sigma Aldrich) and TWEEN-20 (polysorbate 20, available from Sigma Aldrich)). Permeabilized cells may be subjected to one or more washing steps using a buffered aqueous medium.

[0144] The contact of sample with the present composition lasts for a period of time, enough to bind the affinity reagent to the target rare molecule in the sample. This time period can depend on one or more of the properties and sizes of different populations of target rare cells, the properties of the reaction mixture or the volume to be filtered (optional). In some instances, the contact period is from about 1 minute to about 1 hour, from about 5 minutes to about 1 hour, or from about 5 minutes to about 45 minutes, or from about 5 minutes to about 30 minutes, or from about 5 minutes to about 20 minutes, or from about 5 minutes to about 10 minutes, or from about 10 minutes to about 1 hour, or from about 10 minutes to about 45 minutes, or from about 10 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes.

[0145] Determination method

[0146] The composition of the present invention can be used to perform an assay that specifically associates a mass tag with a rare molecule. An exemplary assay uses an affinity reagent to specifically bind to a rare molecule in a sample. The mass tag is associated with the affinity reagent and binds to it. After the capture portion specifically binds to the target analyte, the mass tag is associated with the rare molecule. A separation step is then performed, such as washing away unbound affinity reagents or purifying the rare molecule / affinity reagent / mass tag complex from the sample. After the separation step (washing or purification), the mass tag is then eluted from the affinity reagent by destroying the bond of the tag unit between the affinity reagent and the mass tag using a release agent as described above. The mass tag is then ionized and analyzed. Specifically, an environment that promotes the dissociation of the mass tag from the part can be generated. For example, a pH change can be used to promote dissociation. Alternatively, a chemical reagent can be used to promote dissociation. Alternatively, heat can be used to promote dissociation. In certain embodiments, a combination of techniques is used, such as a combination of pH changes and release agents. Binding and release may involve thiol / disulfide chemistry, which is well established in biochemistry, but alternatives such as complementary binding of PNAs may be used (RJ Ball et al. Artificial DNA: PNA and XNA, 1 (2010) 27-35).

[0147] Specific binding or specific association refers to a binding reaction that determines the target analyte of interest in a heterogeneous population of molecules. Thus, under specified conditions (e.g., immunoassay conditions), a specified capture moiety (e.g., antibody variable domain) binds to its specific "target" and does not bind in large quantities to other molecules present in the sample. Specific binding / association means that the binding is selective in terms of selected target recognition, high, medium, or low binding affinity or avidity. Selective binding is generally achieved if the binding constants or binding kinetics differ by at least 10-fold.

[0148] On the other hand, nonspecific binding involves non-covalent binding between molecules that is relatively independent of specific surface structures. Nonspecific binding can be caused by several factors, including hydrophobic interactions between molecules.

[0149] Mass-tagged ion generation

[0150] Any method known in the art for producing ions of mass labels can be used. Exemplary mass spectrometry techniques for mass spectrometry analysis using an ionization source at atmospheric pressure include electrospray ionization (ESI; Fenn et al., Science, 246: 64-71, 1989; and Yamashita et al., J. Phys. Chem., 88: 4451-4459, 1984); atmospheric pressure ionization (APCI; Carroll et al., Anal. Chem. 47: 2369-2373, 1975); and atmospheric pressure matrix-assisted laser desorption ionization (AP-MALDI; Laiko et al. Anal. Chem., 72: 652-657, 2000; and Tanaka et al. Rapid Commun. Mass Spectrom., 2: 151-153, 1988). The contents of each of these references are incorporated herein by reference in their entirety.

[0151] Exemplary mass spectrometry techniques utilizing direct ambient ionization / sampling methods include desorption electrospray ionization (DESI; Takats et al., Science, 306:471-473, 2004 and U.S. Pat. No. 7,335,897); direct analysis in real time (DART; Cody et al., Anal. Chem., 77:2297-2302, 2005); atmospheric pressure dielectric barrier discharge ionization (DBDI; Kogelschatz, Plasma Chemistry and Plasma Processing, 23:1-46, 2003 and PCT International Publication No. WO2009 / 102766), ion generation using wetted porous materials (Paper Spray, U.S. Pat. No. 8,859,956), and electrospray-assisted laser desorption / ionization (ELDI; Shiea et al., J. Rapid Communications in Mass Spectrometry, 19:3701-3704, 2005). The contents of each of these references are incorporated herein by reference in their entirety.

[0152] Ion generation can be achieved by placing the sample on a porous material and generating mass-tagged ions from the porous material or other types of surfaces, as shown in Ouyang et al., U.S. Patent Application Publication No. 2012 / 0119079, the contents of which are incorporated herein by reference in their entirety. Alternatively, an assay can be performed and ions can be generated from a non-porous material, see, for example, Cooks et al., U.S. Patent Application Serial No. 14 / 209,304, the contents of which are incorporated herein by reference in their entirety). In certain embodiments, a solid needle probe or surface to which a high voltage can be applied can be used to generate mass-tagged ions (see, for example, Cooks et al., U.S. Patent Application Publication No. 20140264004, the contents of which are incorporated herein by reference in their entirety).

[0153] Ion analysis

[0154] In certain embodiments, the ions are analyzed by directing them to a mass spectrometer (benchtop or micro-mass spectrometer). Fig.10is a picture showing various components and their arrangement in a micro mass spectrometer. Control system for Mini 12 (Linfan Li, Tsung-Chi Chen, Yue Ren, Paul I. Hendricks, R. Graham Cooks, and Zheng Ouyang “Miniature Ambient Mass Analysis System” Anal. Chem. 2014, 86 2909-2916, DOI: 10.1021 / ac403766c; and 860. Paul I. Hendricks, Jon K. Dalgleish, Jacob T. Shelley, Matthew A. Kirleis, Matthew T. McNicholas, Linfan Li, Tsung-Chi Chen, Chien-HsunChen, Jason S. Duncan, Frank Boudreau, Robert J. Noll, John P. Denton, Timothy A. Roach, Zheng Ouyang and R. Graham Cooks "Autonomous in-situ analysis and real-time chemical detection using a backpack miniature mass spectrometer: concept, instrumentation development, and performance" Anal. Chem., 2014, 86 2900-2908 DOI: 10.1021 / ac403765x, the contents of each of which are incorporated herein by reference in their entirety) and the vacuum system of Mini 10 (Liang Gao, Qingyu Song, Garth E. Patterson, R. Graham Cooks and Zheng Ouyang, "Handheld Rectilinear Ion Trap Mass Spectrometer", Anal. Chem., 78 (2006) 5994-6002 DOI: 10.1021 / ac061144k, the contents of which are incorporated herein by reference in their entirety) can be combined to produce Fig.10The miniature mass spectrometer shown in . Its size may be similar to a shoe box (H20×W25 cm×D35 cm). In certain embodiments, the miniature mass spectrometer uses a dual LIT configuration, which is described, for example, in Owen et al. (U.S. Patent Application Serial No. 14 / 345,672) and Ouyang et al. (U.S. Patent Application Serial No. 61 / 865,377), the contents of each of which are incorporated herein by reference in their entirety.

[0155] A mass spectrometer (micro or benchtop) can be equipped with a discontinuous interface. Discontinuous interfaces are described, for example, in Ouyang et al. (U.S. Pat. No. 8,304,718) and Cooks et al. (U.S. Patent Application Publication No. 2013 / 0280819), the contents of each of which are incorporated herein by reference in their entirety.

[0156] Factors that should govern the sensitivity of detection are the combined efficiency of all steps in the chemical method, the number of labels per affinity reagent, the number of affinity reagents per rare molecule and the number of ions per mass label, the ionization sensitivity in MS, the S / N ratio in MS, the number of different transitions corresponding to the different mass labels being measured, and the MS acquisition time. However, in such ultratrace analyses, it is often the magnitude of the chemical noise that dominates the performance. It is for this reason that, in the case of complex mixtures, MS / MS often has lower detection limits than normal MS, even though the total signal in the latter case may be orders of magnitude larger.

[0157] Incorporated by Reference

[0158] Other documents, such as patents, patent applications, patent publications, journals, books, papers, and web content, are referenced and cited throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0159] Equivalent

[0160] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, examples, and guidance that can be applicable to the practice of the invention in its various embodiments and their equivalents.

[0161] Examples

[0162] Unless otherwise indicated, the materials used in these examples were from Sigma-Aldrich Corporation (St. Louis MO). Antibody conjugates were prepared by standard bioconjugate techniques, such as those described by Greg T. Hermanson in Bioconjugate Techniques, Third Edition 2013, Elsevier Inc., 225 Wyman Street, Waltham MA. Imidazolium and pyridinium mass markers were prepared by standard imidazolium synthesis techniques, such as those described by Michael W. Pennington and Ben M. Dunn in Imidazolium Synthesis Protocols, First Edition, November 1994, Springer-Verlag, New York, LLC, New York NY. Materials and common terms used in the description of materials are shown in Table 1.

[0163] Example 1: Quadruple mass labeling method for targeting rare molecules

[0164] Cancer cells were prepared for reaction with mass markers by washing cultured SKBR human breast cancer cells twice in 1000 μL phosphate buffered saline (PBS) and centrifuging (2 minutes, 2500 rpm (Eppendorf Centrifuge 5417C)). The solution was discarded both times to obtain a cell pellet. As a permeabilization step, the cells were suspended in 1000 μL PBS containing 0.2% Triton X100 (PBST 0.2%), the cells were incubated for 7 minutes, and then washed 4 times in 1000 μL PBS. As a blocking step, the cells were suspended in 1000 μL casein buffer, the cells were incubated for 7 minutes, and then washed once in 1000 μL PBST0.05%). The cells were reacted with 300 μL of 10 μg / mL CK-8-18 antibody conjugated with Dylight 550 fluorescent marker in casein buffer to visualize the cancer cells. The cells were incubated for 25 minutes and then washed 4 times in 1000 μL PBST (0.05%). The cells were reacted with 500 μL of 1 μg / mL 4,6-diamidino-2-phenylindole (DAPI) solution to visualize the cancer cell nuclei. The cells were incubated for 1 minute and then washed 4 times in 1000 μL PBST (0.05%). The stained cell mixture was diluted to 1 mL with PBS and 2 μL samples were examined under a microscope to verify the stained cell count (~66500 cells / mL).

[0165] The mass tag was prepared by coupling L-carnitine-FGGSC (QC5-1) and biotin to propylamine-functionalized silica and a cleavable disulfide linker (i.e., tag binding unit) using a coupling agent, 3-(2-pyridyldithio) propionic acid N-succinimidyl ester (SPDP). Antibodies against Her-2 / neu rare molecules were conjugated with Dylight488 and further conjugated to propylamine-functionalized silica (nanoparticles, 200 nm, mesopore size 4 nm) via an amide bond with N-hydroxysuccinimidyl ester to prepare antibody-nanoparticles (affinity reagents, Figure 1 ). Biotin was disulfide bonded to the affinity reagent via SPDP reaction. QC5-1 nanoparticles and affinity reagent were conjugated to streptavidin magnetic beads (Pierce 1%, 0.756 μM, 10 mg / ml) to provide 18.2 ug / mL of combined antibody-QC5-1 particles to prepare a quadruple mass tag attached to the affinity reagent. Other mass tags were attached to the affinity reagent using the same method.

[0166] The stained cells were reacted with the quadruple mass tag attached to the affinity by adding 300 μL of 0.075 μg / ml antibody-QC5-1 particles to the stained SKBR3 cells (~20,000 cells). The resulting mixture was incubated at 75 rpm on a roller mixer for 2 hours at room temperature. The mixture was centrifuged, the liquid was decanted, and then washed twice with 1 mL PBST (0.3%) and twice with 1 mL PBS. The reacted cells were examined under a fluorescent microscope and it was verified that 100% of the SBKR cells reacted with the antibody-QC5-1 particles. This demonstrates that the quadruple mass tag method can be used for rare molecule detection.

[0167] Example 2: Detection of four-level quality markers

[0168] A benchtop linear ion trap mass spectrometer (LTQ Thermo Fisher) was used to analyze mass-labeled ions generated by nanoelectrospray ionization (nESI) to test the detection limit (estimated concentrations giving a signal-to-noise ratio equal to 3). The instrument settings were as follows: spray voltage, 1.5 kV; capillary temperature, 150 °C; tube lens, 65 V; capillary voltage, 15 V; collision energy, 35; Q activation, 0.3.

[0169] A list of compounds tested is provided in Table 1, which includes types of potential mass markers with and without quaternary structure, and includes different 3 amino acid peptides, 6 amino acid peptides, and 9 amino acid peptides as well as small molecule quaternary imidazolium salts (methylimidazolium-methyl-salicylaldehyde, MIMSA) and quaternary staining reagent Nile Blue A. For peptides, quaternary structure was prepared by adding an L-carnitine residue to the N-terminus. Peptides lacking quaternary structure were detected at a detection limit of 10 to 50 nM. Adding quaternary structure increased the detection limit to 1 nM. This indicates that quaternary mass markers allow for higher sensitivity and are easily ionized for detection.

[0170] Table 1: Mass marker detection limits of nESI

[0171]

[0172] Example 3: Quaternary Mass Labeling of Methylimidazolium and Pyridinium Analogs

[0173] Synthesis of quaternary mass-tagged methylimidazolium and pyridinium analogs. To attach such mass tags to propylamine-functionalized silica nanoparticles, either imines were formed directly or the nanoparticles were modified with 1,2-diols to form acetals through the aldehyde functionality of the quaternary mass tags. Methylimidazolium and pyridinium analogs were prepared from salicylaldehyde by first performing a Blanc chloromethylation reaction, followed by isolation of the product and further reaction with N-substituted imidazole or pyridine analogs. Changes in the structure of the N-substituent of the imidazole or substitution of the pyridine introduced into the chloromethylated salicylaldehyde facilitated the synthesis of mass tags with different structures. Figure 5 Methylimidazolium (m / z 217) and pyridinium (m / z 214) mass markers are shown. The mass markers fragment upon ionization to produce unique product ions that are subsequently analyzed by MS / MS and are represented by methylimidazolium (m / z 83) and pyridinium (m / z 80).

[0174] Figure 6 Shows how an affinity reagent or rare molecule represented by R is attached to a quaternary mass marker via an acetal or ketal bond. The bond is formed by the reaction of a diol attached to R and an aldehyde of the quaternary mass marker. The diol is first attached to the amine of the affinity reagent or rare molecule (R) via the reaction of β-hydroxy-γ-butyrolactone to form an amide bond. The detailed procedure for making such a material is as follows:

[0175] Preparation of Chloromethyl Salicylaldehyde

[0176] At room temperature, paraformaldehyde (1 g) was suspended in 12M hydrochloric acid (4 mL) under stirring. The paraformaldehyde powder was partially dissolved in acid (so that no dried paraformaldehyde floated on the liquid), followed by salicylaldehyde (0.6 g was added dropwise over a half hour process). The resulting mixture presented a yellow hue and was finally clarified when the paraformaldehyde was completely dissolved. The solution was stirred for 12 hours, and it lost color over time, forming a white precipitate-shaped product. The product was filtered and washed three times with deionized water to remove residual acid. The crude material was air-dried and used for the next step without further purification.

[0177] Preparation of (Methylimidazole)methylsalicylaldehyde

[0178] Crude chloromethyl salicylaldehyde (1 eq) was dissolved in dichloromethane at room temperature. Methylimidazole (1.5 eq) was added dropwise, causing the desired product to precipitate almost immediately (in small portions when imidazole was added). The precipitate was filtered and washed three times with dichloromethane to remove all residual organics (substantially purifying the material).

[0179] General procedure for forming imine-bound nanoparticle conjugate species

[0180] Methylimidazolium)methyl salicylaldehyde (1.2 eq) was dissolved in anhydrous methanol (1 mL) in an Eppendorf tube. The corresponding amine conjugate partner (1 eq) was added to the Eppendorf tube and the solution was left for 30 minutes, during which time it developed a yellow color, indicating the formation of the imine.

[0181] Preparation of acetal-protected imidazolium mass markers

[0182] The imidazolium mass marker (0.3 g) was dissolved in anhydrous dimethylformamide and Amberlyst-15 (hydrogen form, mass not determined) was added. Once the imidazolium mass marker was dissolved, ethylene glycol (0.2 g) was added to the solution and the reaction mixture was stirred for 6 hours. The structure of the resulting acetal was confirmed by mass spectrometry.

[0183] Preparation of diol-functionalized propylamine

[0184] Propylamine (neat, 1 equivalent) was mixed with β-hydroxy-γ-butyrolactone (neat, 1 equivalent). As the reaction proceeded to completion, the reaction immediately exothermed and eventually crystallized. The yield was essentially quantitative.

[0185] Silica nanoparticles functionalized with diol-modified propylamine

[0186] Propylamine functionalized silica nanoparticles (0.03 g) were overlaid with β-hydroxy-γ-butyrolactone (300 μL) in an Eppendorf tube and allowed to react for 1 hour. On a larger scale, the reaction produced an exotherm strong enough to be felt on the skin, indicating that the amidation reaction was similar to that observed for free propylamine.

[0187] Figure 5 Quaternary mass labeled imidazolium (A) and pyridinium (B) analogs prepared by this method are shown. Methylimidazolium (m / z 217) and pyridinium (m / z 214) mass labels. In these examples, the mass structure consists of all other atoms that are not directly connected to the formal positive charge by a single bond or by conjugation, namely the salicylaldehyde moiety and the methyl group. The aldehyde of methylsalicylaldehyde is used to bind the amine of the propylamine functionalized silica nanoparticles through an imine bond or, after modification of the amine, through an acetal bond, as shown in Figure 3.

[0188] Two methods of attachment to aldehydes were compared. Imine bonds (Inset D of Figure 3) and acetal bonds (Inset B of Figure 3) were prepared using organic amines as models for rare molecules and affinity reagents. Conjugates of MIMSA with propylamine, mercaptoaniline and 1,6-diaminonaphthalene were studied. The imine bond forms rapidly in anhydrous methanol. It was found that the bond is easily cleaved in the presence of aqueous solvents such as PBS. In order to use these materials in the method, it is important that the bond used to connect the quaternary mass marker is stable in the buffered aqueous solution commonly used for the assay procedure. The imine system was considered unacceptable because the bond was completely hydrolyzed after 20 minutes of exposure to aqueous solvents. The acetal and material were almost completely stable for at least 20 minutes.

[0189] To conjugate with organic amines via acetal bonds, the free amines were first functionalized with 1,2-diol structures by reaction with β-hydroxy-γ-butyrolactone (see Figure 6 ). This reaction scheme was successfully applied to organic amines and amine nanoparticles. It produced the desired diol structure (labeled binding unit, Figure 1 ), and showed an effective method for conjugation to mass-tagged aldehydes and ketones. The resulting acetals and ketals have the same hydrolytic stability as ethylene glycol-derived acetals. Acetals and ketals are also easily separated from affinity reagents by a simple one-step liquid addition of an acidified spray solvent such as 50% acetonitrile in water and 0.1% trifluoroacetic acid (TFA). No suppression of mass marker signals in the mass spectra was observed by nESI from 50% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA). In contrast, the use of tris(2-carboxyethyl)phosphine hydrochloride) at 1 mg / mL in water to disrupt disulfide bonds resulted in a 50% or more suppression of mass marker detection limits by nESI. Therefore, disulfide bonds are not suitable for attaching quaternary mass markers.

[0190] An example workflow incorporating all of the above elements was performed. Propylamine functionalized silica nanoparticles were modified with β-hydroxy-γ-butyrolactone. A mass-labeled methylimidazolium analog ( Figure 5 , A) is bound to the resulting diol-functionalized nanoparticles. After this conjugation and subsequent washing steps, the mass marker bound to the nanoparticles is released into a release solution of aqueous trifluoroacetic acid and the released mass marker is quantified using a calibration curve against an internal standard. The internal standard is a mass-labeled pyridinium analog ( Figure 5 , B). The calculated mass marker concentration in the release solution was two orders of magnitude higher than the concentration measured from the supernatant of the previous wash step ( Figure 7 ).

[0191] Figure 8 The mass tags with high molecular weight derivatives of imidazolium (m / z 309 and 259) were shown to effectively form mass tags with higher mass variable mass units. These mass tags were also fragmented to produce unique product ions (m / z 175 and m / z 125) that were predictable and separable from background mass spectrometry.

Claims

1. A method for detecting a target analyte in an environmental sample, or a food or beverage product, the method comprising: introducing a charged mass marker composition into an environmental sample, or a food or beverage product, comprising a target analyte, wherein the charged mass marker composition comprises an affinity reagent; and a mass marker precursor bound to the affinity reagent, wherein the mass marker precursor comprises a marker binding unit and a mass marker, the marker binding unit reversibly binding the mass marker to the affinity reagent, and wherein the mass marker comprises a charge unit chemically coupled to a mass marker unit, the mass marker unit being neutral and having a predefined mass-to-charge ratio value in a mass spectrum; in, Upon ionization, the charge unit is released from the mass marker unit and the mass marker unit is detectable in the mass spectrum due to the mass marker unit having a predefined mass-to-charge ratio value in the mass spectrum, and wherein the predefined mass-to-charge ratio value in the mass spectrum of the mass marker or one or more fragments of the mass marker is different from a background mass-to-charge ratio value in the mass spectrum; incubating the environmental sample, or food or beverage product and the charged mass marker composition so that the charged mass marker composition binds to the target analyte through an interaction between an affinity agent of the charged mass marker composition and the target analyte; releasing the mass tag from the affinity reagent by breaking one or more bonds of the tag binding unit; ionizing the mass marker; and The ionized mass tags and / or fragments thereof are detected in a mass spectrometer, thereby detecting the target analyte in the environmental sample, or food or beverage product. 2 . The method according to claim 1 , further comprising quantifying the ionized mass tag and / or fragments thereof, thereby quantifying the target analyte in the sample.

3. The method of claim 1, wherein the mass label unit and the charge unit are bound to each other via one or more bonds that are resistant to breakage upon ionization. The method of claim 1 , wherein the label binding unit comprises a reversible bond. The method of claim 4 , wherein the reversible bond comprises an acetal or ketal bond.

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