JP2008503738A - Analysis method - Google Patents

Abstract

  A method for quantitative measurement using mass spectrometry of one or more peptides generated from one or more analyte proteins by enzymatic or chemical cleavage, wherein the one or more mass labeled peptide internal standard precursors Exposing together a sample to be analyzed and one or more mass label peptide internal standard precursors to enzymatic or chemical cleavage to produce one or more mass label peptide internal standards from Wherein the mass-labeled peptide internal standard precursor comprises an N-terminal extension of at least one amino acid relative to the most N-terminal N-terminus of the mass-labeled peptide internal standard resulting from the precursor, and the mass resulting from the precursor A C-terminal extension of at least one amino acid relative to the most C-terminal C-terminus of the label peptide internal standard.

Description

  The present invention provides methods and reagents for quantifying proteins and peptides from complex polypeptide mixtures such as plasma, tissue, and cell culture.

  Protein and peptide concentration levels in endogenous samples such as plasma and tissues are usually measured by immunochemical techniques such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). These methods are well established, sensitive and usually very reliable, but require access to specific antibodies and are associated with cross-reactivity, low reproducibility, and epitope denaturation You can be troubled by problems. In addition, ELISA and RIA assays are typically developed to analyze one analyte per assay and are not well suited for complex analyses.

  Mass spectrometry (MS) is an established method for quantification of low molecular weight compounds in complex samples such as plasma, often combined with high performance liquid chromatography. The main advantages of detection by mass spectrometry include high resolution and high sensitivity, which allows absolute quantification of low levels of analytes in complex samples.

  Development of “soft ionization” techniques such as matrix-assisted laser desorption ionization (MALDI) and electrospray has enabled the analysis of large biomolecules such as peptides and proteins using mass spectrometry. MS is mainly used for qualitative analysis such as sequence analysis, protein identification, and analysis of post-translational modifications.

  As a means of quantification, MS is used in a manner similar to that for low molecular weight compounds, ie combined with high performance liquid chromatography or immunoaffinity chromatography to generate internal, mass-shifted standard peptides. The detection method used has been used to quantify smaller peptides (references 1-6). Protein quantification is not easy because one of the reasons is that the resolution of intact proteins allows the different protein species in the complex sample to be resolved or the analyte. This is because it is usually insufficient to distinguish between the signal of and the internal standard signal.

  Semi-quantification applications include surface-enhanced laser desorption / ionization (SELDI) (Ref. 7), which extracts protein sub-fractions from complex samples by affinity surfaces and uses linear MALDI-TOF. The analysis is performed by mass spectrometry. Occasionally, changes in the relative abundance of individual proteins may be detected by comparing spectra generated from different samples (eg, plasma from healthy / disease patients).

  Another approach for measuring protein concentration is to fragment the protein into shorter peptides by enzymatic or chemical means. The advantage of analyzing peptides rather than unprocessed proteins is that peptides exhibit more homogeneous physiochemical properties and can be analyzed by high resolution mass spectrometry techniques such as reflectron MALDI-TOF MS or electrospray MS It tends to fall within the molecular weight range. On the other hand, sample complexity increases because a single protein yields many different peptides. Thus, protein analysis in complex samples by fragmentation usually requires a high resolution separation step prior to MS analysis to reduce the number of analytes introduced into the mass spectrometer. Such pre-fractionation of a fragmented sample can include multidimensional liquid chromatography and / or extraction of peptide subgroups by affinity tags.

  One method for quantifying a given peptide derived from a particular protein is to use an internal standard with the same amino acid sequence but with a mass shift obtained by chemical modification or by introduction of a stable isotope. Is to add. As an example, Barr et al. Demonstrated a method for measuring apolipoprotein A-1 by trypsin fragmentation and addition of an isotope-labeled internal standard peptide (Reference 8). Recently, the same approach has been described by Gerber et al. (Ref. 9), but in this study MS / MS was used for quantification. This work by Gerber et al. Is also the subject of WO 03/016861. Gerber et al. Describe two “ragged ends” and reactive amino acids, if possible, should be avoided by the choice of protease and peptide, but without two adjacent endoprotease cleavage sites (unmatched ends). If the peptides they have to be analyzed, it is taught that an N-terminal extension can be added to explain which cleavage site is dominant.

  Another variation is to add an affinity label such as biotin to a subpopulation of peptides, for example by using cysteine specific chemical modifications. By introducing affinity tags with different masses, it is possible to measure the relative difference in protein content between two different samples. This method is called the isotope-coded affinity tag (ICAT) (Ref. 10) method and has the advantage of introducing a mass shift into the analyte in the same step as adding the affinity tag. However, this method will not result in absolute quantification of the protein or peptide of interest.

Preferably, the internal standard should be added to the sample to be analyzed as early as possible during the analytical process so that all sample manipulation steps equally affect the internal standard and the analyte. . The addition of synthetic small peptides to the protein mixture prior to sample preparation can lead to several problems: i) The yield of the internal standard after sample preparation will not reflect the efficiency of the fragmentation process; ii) Small, possibly hydrophobic peptides can be partially or completely lost during sample preparation due to adsorption to other protein or plastic sample containers; and iii) small peptides can precipitate, limit During certain sample preparation steps such as external filtration and chromatography, it may not necessarily be co-purified with the protein to be analyzed.
International Publication No. 03/016861 Pamphlet Barr JR, et al., "Isotope dilution-mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I." Clin Chem. 1996 Oct; 42 (10): 1676-82

  Therefore, there is a need for protein and peptide quantification methods that do not have the disadvantages described above.

[Summary of the present invention]
The present invention provides a method for quantitative measurement of peptides produced from unprocessed proteins by enzymatic or chemical cleavage using a mass labeled peptide internal standard precursor. The mass label peptide internal standard precursor contains an amino acid sequence that is identical or nearly identical to the protein fragment to be analyzed (eg, one amino acid is different) but extends with one or more amino acids at both the C-terminus and N-terminus Has been.

  The present invention also provides kits that may be used to quantify proteins and peptides in a sample.

  The invention also provides a diagnostic method for quantifying one or more peptides or proteins in a sample.

[Detailed Description of the Invention]
The term “mass-labeled peptide internal standard precursor” includes synthetic peptides or recombinants that contain one or several amino acid sequences corresponding to one or several known or predicted protein sequences Proteins are included, which, when fragmented by proteases or chemicals, the internal standard precursor is identical or nearly identical to the fragments resulting from one or more proteins to be analyzed, except for slightly different molecular weights. Labeled in such a way as to produce a fragment. The chemical modification of the mass label peptide internal standard precursor is preferably the incorporation of a stable isotope. In this case, the fragment from the internal standard precursor and the fragment from the protein to be analyzed will be identical, except for a slightly different molecular weight.

  The term “polypeptide” includes compounds comprising two or more amino acids joined by peptide bonds.

  “Protein” means any protein without limitation, and preferably includes at least 50 amino acids.

  “Peptide” means a shorter polypeptide, preferably including 50 or fewer amino acids, such as those containing 4 to 50 amino acids or 10 to 24 amino acids.

  “Analyte protein” and “analyte peptide” mean a specific protein or peptide to be quantified.

  A first aspect of the present invention is a quantitative measurement method using mass spectrometry of one or more peptides generated from one or more analyte proteins by enzymatic or chemical cleavage, In order to generate one or more mass label peptide internal standards from one or more mass label peptide internal standard precursors, the sample to be analyzed and one or more mass label peptide internal standard precursors, Providing a method comprising exposing together to enzymatic or chemical cleavage, wherein the mass label peptide internal standard precursor is relative to the N-terminal of the most N-terminal side of the mass label peptide internal standard resulting from the precursor An N-terminal extension of at least one amino acid and a C-terminus of at least one amino acid relative to the most C-terminal C-terminus of the mass label peptide internal standard resulting from the precursor And a length portion.

  In an embodiment of this aspect of the invention, the N-terminal extension is the protein to be analyzed (before cleavage) in the sequence of the protein corresponding to the most N-terminal side of the mass-labeled peptide internal standard derived from the precursor. An amino acid sequence of at least 1, 2, 3, 4, or 5 amino acids identical to the existing amino acid sequence adjacent to the N-terminal sequence is included at the C-terminal end of the extension.

  In a further embodiment of this aspect of the invention, the C-terminal extension is the protein to be analyzed (prior to cleavage) in the sequence of the protein to be analyzed corresponding to the most C-terminal side of the mass-labeled peptide internal standard derived from the precursor. A) an amino acid sequence of at least 1, 2, 3, 4, or 5 amino acids identical to the amino acid sequence adjacent to the C-terminal sequence present is included at the N-terminal end of the extension.

  In a further embodiment of this aspect of the invention, the amino acid sequence of at least 1, 2, 3, 4, or 5 amino acids adjacent to the N-terminus of the mass-labeled peptide internal standard resulting from the precursor is the mass The sequence of the protein to be analyzed corresponding to the sequence of the label peptide internal standard is identical to the amino acid sequence adjacent to the N-terminal sequence present in the protein to be analyzed (before cleavage).

  In a further embodiment of this aspect of the invention, the amino acid sequence of at least 2, 3, 4, or 5 amino acids adjacent to the C-terminus of the mass label peptide internal standard resulting from the precursor is The sequence of the protein to be analyzed corresponding to the standard sequence is identical to the amino acid sequence adjacent to the C-terminal sequence present in the protein to be analyzed (before cleavage).

  The present invention provides a method for quantitative measurement of peptides produced from unprocessed proteins by enzymatic or chemical cleavage using a mass labeled peptide internal standard precursor. The mass label peptide internal standard precursor comprises the same or nearly the same amino acid sequence as the protein fragment to be analyzed, but is extended with one or more amino acids at both the C-terminus and the N-terminus. This extension may or may not be identical to the amino acid extension of the protein to be analyzed adjacent to the resulting fragment prior to fragmentation. Cleavage of the analyte protein and the mass label peptide internal standard precursor yields an analyzable peptide from the protein and an internal standard peptide from the internal standard precursor, and from an analytical point of view, the only difference between the two is Typically, only a 2-10 Dalton mass shift (FIG. 1).

  The use of a mass-shifting peptide internal standard precursor provides several advantages: i) Variations in peptide yield after fragmentation of one or more proteins can be determined by whether the internal standard precursor is an analyte protein or analyte. Ii) The internal standard precursor is analyzed during the various sample preparation steps that take place prior to protein fragmentation. And iii) a single internal standard precursor can be used to analyze multiple internal standard peptides to analyze peptide fragments from several different analyte proteins or peptides. Can be designed to generate.

Mass label peptide internal standard precursors can be made by peptide synthesis, during which one or more mass label amino acids are incorporated. Preferably, the mass labeled amino acids include one or more stable isotopes including but not limited to ¹³ C, ¹⁵ N, ¹⁸ O, ² H, and ³⁴ S.

  Peptides may be synthesized by the Fmoc-polyamide method of solid phase peptide synthesis, as disclosed in Lu et al (1981) J. Org. Chem. 46, 3433, incorporated herein by reference. . Temporary N-amino group protection is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. This repeated cleavage of the high base-labile protecting group is performed using 20% piperidine in N, N-dimethylformamide. The side chain functional groups are butyl ether (for serine, threonine and tyrosine), butyl ester (for glutamic acid and aspartic acid), butyloxycarbonyl derivative (for lysine and histidine), trityl derivative (for cysteine), and It may be protected as a 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivative (in the case of arginine). When glutamine or asparagine is the C-terminal residue, a 4,4'-dimethoxybenzhydryl group is used for protection of the side chain amide functionality. The solid support is based on a polydimethylacrylamide polymer consisting of three monomers: dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (crosslinking agent), and acryloyl sarcosine methyl ester (functionalizing agent). An acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative is used as the cleavable linking agent between the peptide and the resin. All amino acid derivatives, except for asparagine and glutamine, are added as their preformed symmetrical anhydride derivatives, which are coupled with N, N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole. It is added using the reverse procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulfonic acid, or isotine test methods. Upon completion of the synthesis, treatment with 95% trifluoroacetic acid containing 50% scavenger mix removes side chain protecting groups and cleaves the peptide from the resin support. Commonly used scavengers are ethanedithiol, phenol, anisole, and water, which are appropriately selected according to the constituent amino acids of the peptide to be synthesized. Trifluoroacetic acid is removed by vacuum evaporation, followed by trituration with diethyl ether to give the crude peptide. Any scavenger present is removed by a simple extraction method in which a crude peptide free of scavenger is obtained by lyophilization of the aqueous phase. Peptide synthesis reagents are usually available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be performed by any one or a combination of techniques such as size exclusion chromatography, ion exchange chromatography, and (primarily) reverse phase high performance liquid chromatography. Peptide analysis may be performed using thin layer chromatography, reverse phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, and fast atom bombardment (FAB) mass spectrometry.

  A mass label may also consist of other chemical modifications of amino acids, including but not limited to acetylation, methylation, deamidation, and carboxymethylation. Where mass label modifications are other than stable isotope incorporation, it is important to avoid modifications that may alter internal standard peptide behavior during sample preparation, separation, and ionization.

  In addition, the mass label peptide internal standard precursor may be synthesized to have one or more substituted amino acid residues associated with the protein or peptide to be analyzed: for example, replacement of glycine with alanine. Under such circumstances, cleavage of the protein or peptide to be analyzed and the mass-labeled peptide internal standard precursor yields an analyzable peptide from the protein, and an internal standard peptide from the internal standard precursor. From an analytical point of view, The only difference between them is a mass shift due to the presence of alanine in the peptide internal standard relative to the presence of glycine in the analyte protein or peptide. An advantage of such an embodiment of the present invention is that during the synthesis of a mass-labeled peptide internal standard precursor, it may be easier to replace alanine with glycine rather than with isotope-labeled alanine. is there. One skilled in the art can readily appreciate additional suitable amino acid substitutions, for example, valine and isoleucine may be exchanged.

  The mass label peptide internal standard precursor may have 0, 1, 2, 3, 4, or 5 substituted amino acid residues associated with the protein or peptide to be analyzed, or the analyte In relation to proteins or peptides, up to 5%, 10%, 15%, 20%, or 25% of the total number of amino acid residues may be different. Preferably, the mass label peptide internal standard precursor has no substituted amino acid residues or no more than one substituted amino acid residue in relation to the analyte protein or peptide.

  If the protein fragment to be analyzed contains any post-transcriptional modification such as phosphorylation, the mass label peptide internal standard may be synthesized such that the same modification is made.

  Mass-labeled peptide internal standard precursors may also be produced by recombinant protein expression in the presence of mass-labeled amino acids as part of the hybrid protein, where such hybrid proteins constitute internal standard precursors. Or the hybrid protein may be processed to produce one or more peptide internal standard precursors.

  When preparing mass-labeled peptide internal standard precursors using recombinant protein expression, Sambrook et al (2001) “Molecular Cloning, a Laboratory Manual”, 3rd edition, Sambrook, is well known to those skilled in the art. A DNA molecule encoding the desired peptide is prepared using the method illustrated by et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

  The DNA is then expressed in a suitable host to produce a mass labeled peptide internal standard precursor. Accordingly, it has been appropriately modified in view of the teachings contained herein to construct an expression vector and then use it to transform a host cell suitable for expressing and producing the peptide. According to a known technique, DNA encoding the peptide may be used. Such techniques include those disclosed below: U.S. Pat. No. 4,440,859, acquired April 3, 1984 by Rutter et al .; the same, acquired July 23, 1985, by Weissman. No. 4530901; Acquired by Crowl on April 15, 1986 No. 4582800; Acquired by Mark et al. On June 30, 1987 No. 4677063; Acquired by Goeddel on July 7, 1987 No. 4,678,751; No. 4,704,362 acquired on November 3, 1987 by Itakura et al .; No. 4,710,463 acquired on December 1, 1987 by Murray; July 1988 by Toole, Jr. et al. No. 4775706, acquired on 12th; No. 4766075, acquired on 23 August 1988 by Goeddel et al .; and 1989 by Stalker. No. 4,810,648, acquired on March 7, 2013, all of which are incorporated herein by reference.

  DNA encoding the mass label peptide internal standard precursor may be combined with a wide variety of other DNA sequences for introduction into a suitable host. The accompanying DNA will depend on the characteristics of the host, how the DNA is introduced into the host, and whether episomal retention or integration is desired.

  Usually, the DNA is inserted into an expression vector such as a plasmid, with the proper arrangement for expression and the correct reading frame. If necessary, the DNA may be combined with appropriate transcriptional and translational control control nucleotide sequences that are recognized by the desired host, although such controls are usually available in expression vectors. . Thus, the DNA insert may be operably linked to a suitable promoter. Bacterial promoters include E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, phage λPR and PL promoters, phoA promoter, and trp promoter. Eukaryotic promoters include CMV early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, and retroviral LTR promoter. Other suitable promoters are known to those skilled in the art. The expression construct also preferably includes transcription initiation and termination sites, as well as a ribosome binding site for translation in the transcription region (Hastings et al., WO 98/16643, published April 23, 1998). issue).

  The vector is then introduced into the host through standard techniques. Usually, not all of the host is transformed by the vector, so it will be necessary to select transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence marker that encodes a selectable trait in the transformed cell, along with any necessary regulatory elements. These markers include dihydrofolate reductase, G418 resistance, or neomycin resistance for eukaryotic cell culture, and tetracycline resistance gene, kanamycin resistance gene, or ampicillin for culture in E. coli and other bacteria. Resistance genes are included. Alternatively, the gene for such a selectable trait can be placed in another vector, which is used to co-transform the desired host cell.

  The host cells transformed with the recombinant DNA encoding the mass label peptide internal standard precursor are then cultured for a sufficient amount of time under appropriate conditions known to those skilled in the art to allow expression of the polypeptide and then recovered. it can.

  Mass labeled peptide internal standard precursors can be recovered and purified from recombinant cell cultures by well-known methods including the following methods: ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic Interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

  Many expression systems are known, including systems using the following: bacteria transformed with recombinant bacteriophages, plasmids, cosmid DNA expression vectors, etc. (eg, E. coli and Bacillus subtilis, etc.); Yeast transformed with a yeast expression vector or the like (eg, Saccaromyces cerevisiae); eg, an insect cell system transformed with a viral expression vector (eg, baculovirus); eg, transfected with a viral or bacterial expression vector, etc. Plant cell lines; animal cell lines transfected with, for example, adenovirus expression vectors.

  Amino acid sequences suitable for use as mass label peptide internal standards predict protein and fragmented products of proteins based on the specificity of the protein and the amino acid sequence of the protein to be analyzed using in silico methods well known to those skilled in the art May be determined. One or more amino acid sequences suitable for use as peptide internal standard precursors may also be determined by fragmentation and mass spectrometry of the protein to be analyzed to determine the actual primary structure of the resulting peptide fragment. In another aspect, for example, unknown peptides discovered by analyzing differences in fragmented proteins from healthy patients, sick patients, etc. are identified, sequenced, and the information is mass labeled peptide It may be used to design an internal standard precursor. In this case (as an example), the extension sequence may not match the flanking sequence of the protein to be analyzed, provided that an unknown peptide can identify the polypeptide derived from it (after sequencing). ), Thereby excluding where it is possible to identify the flanking amino acids of the protein to be analyzed.

  The protein to be analyzed can be any known protein or hypothetical protein predicted by analysis of nucleic acid sequences. The protein to be analyzed was also not previously known and its presence used the following methods: two-dimensional electrophoresis, liquid chromatography, mass spectrometry, or other analytical methods, or a combination thereof It can also be suggested by protein expression analysis of unprocessed protein or fragmented protein. Such methods are well known to those skilled in the art.

  Mass-labeled peptide internal standard precursors may be designed to detect and measure modified proteins and peptides, where the modifications include but are not limited to phosphorylation, glycosylation, oxidation, farnesylation, acetyl , Ubiquitination, lipidation, prenylation, and sulfonation. These are also known or predicted proteins and peptides produced by alternative splicing of mRNA, by specific or non-specific degradation of proteins in vivo, or by mutations resulting from single nucleotide polymorphisms It may be designed to detect and measure species.

  A single internal standard precursor may be designed and synthesized to yield one, two, or more internal standard peptides upon cleavage. Accordingly, in a further embodiment of this aspect of the invention, the mass label peptide internal standard precursor comprises two or more mass label peptide internal standards. In a further embodiment of this aspect of the invention, the mass label peptide internal standard precursor comprises a plurality of mass label peptide internal standards to analyze peptide fragments derived from a plurality of analyte proteins.

  The heterogeneous sample of peptide or protein may be extracted from a cell or tissue sample, or typically (but not necessarily) a peptide and It may be derived from fragmentation of a heterogeneous sample of protein. The cell or tissue sample may be derived from normal or diseased tissue. A cell or tissue sample may be derived from tissue in various differentiated or active states. Further suitable sources of proteins and peptides include tissue material from prokaryotes, eukaryotic cell lines, knockout mice and other animal models, and transgenic plants and plant material. Methods for extracting proteins from such tissue samples are well known to those skilled in the art.

  The mass label peptide internal standard used in this aspect of the invention can be of any length suitable for the method of the invention, typically 4-50 amino acids long, preferably 10-40 amino acids long. is there. The size of the mass-labeled peptide internal standard depends on the requirement for the standard peptide to have a minimum size that can be related to the analyte protein or peptide and a maximum size that can be used to separate the standard peptide using conventional mass spectrometry. It is decided.

  In a further embodiment of this aspect of the invention, the mass labeled peptide internal standard precursor has a length of 6-200 amino acids. As mentioned above, the precursor may comprise one or several amino acid sequences corresponding to the sequence of one or several known or predicted proteins. Since the mass label peptide internal standard precursor is extended with one or more amino acids at both the C-terminus and the N-terminus, the minimum size of the mass label peptide internal standard precursor is 6 amino acids.

  In a further embodiment of this aspect of the invention, the mass labeled peptide internal standard precursor is one or more analytes to be measured during the sample preparation step performed prior to exposure to enzymatic or chemical cleavage. Co-purified with analytical protein.

  In a further embodiment of this aspect of the invention, the mass labeled peptide internal standard precursor is combined with the sample to be analyzed, after any sample collection, but prior to any sample preparation or fragmentation step.

  With reference to FIGS. 2 and 3, the mass shift peptide internal standard precursor is preferably added to the sample to be analyzed immediately after sample collection, preferably prior to any sample preparation or fragmentation step. Thus, the mass shift peptide internal standard precursor and the protein or peptide to be analyzed undergo the same sample preparation or fragmentation step. Several different internal standard precursors may be added to allow quantification of several different proteins and peptides simultaneously, and several internal standard precursors may be added to obtain redundant information. It can be added to different fragments from the same protein. Alternatively, a single internal standard precursor that yields more than one internal standard may be used.

  An analytical sample can then be prepared, if necessary, to remove substances that may interfere with the analysis and to enrich one or more analytes. Methods that may be used include solid phase extraction, liquid chromatography, precipitation, ultrafiltration, and purification using affinity-based techniques, as will be understood by those skilled in the art.

  The protein in the sample may also be chemically modified, such as by cysteine reduction and carboxymethylation to cleave disulfide bonds and avoid formation of peptide dimers.

  The method of the invention includes a step of fragmenting a heterogeneous sample of proteins or peptides to produce a heterogeneous sample of peptide fragments.

  Fragmenting a heterogeneous sample of protein, polypeptide, or peptide may be accomplished by any method known in the art. For example, chemical or enzymatic cleavage may be used. Numerous methods of chemical or enzymatic (ie, protease directed) cleavage are known in the art. For example, proteases include trypsin, chymotrypsin, pepsin, thrombin, papain, bromelain, thermolysin, subsilisin, factor Xa, Staphylococcus aureus protease, and carboxypeptidase A. In preferred embodiments, the fragmentation method cleaves a protein, polypeptide, or peptide at a defined location. Enzymatic cleavage is typically sequence-directed as shown in Table 1 below. Thus, in a further embodiment of this aspect of the invention, the enzymatic or chemical cleavage is sequence directed.

  Chemical cleavage methods can also be sequence-directed, such as cyanogen bromide fragmentation, which cleaves proteins or peptides on the C-terminal side of methionine.

In the table, R ₁ and R ₂ are represented by the following formula:
N-terminal NH—CHR ₁ —CO—NH—CHR ₂ —CO C-terminal is defined.

  Thus, for example, trypsin cleavage is guided by the presence of an arginine or lysine residue in the protein, polypeptide, or peptide, and thus has either arginine or lysine as its C-terminal residue. It is a sequence-directed fragmentation tool because it yields a cleaved fragment.

  As described above, the mass label peptide internal standard precursor comprises an N-terminal extension of at least one amino acid relative to the most N-terminal N-terminus of the mass label peptide internal standard resulting from the precursor, and the mass label peptide internal standard resulting from the precursor A C-terminal extension of at least one amino acid relative to the standard C-terminal C-terminus. Suitable amino acid residues for such elongation include hydrophilic or neutral amino acids such as, for example, glycine, alanine, valine, leucine, isoleucine, serine, and threonine.

  Less suitable amino acid residues include cysteine (which can induce disulfide bond formation), proline (which does not give the resulting peptide much flexibility, affects the structure of the epitope and is sensitive to trypsin cleavage) Low peptide), and hydrophobic or aromatic residues, which can reduce the solubility of the peptide. Furthermore, if trypsin is used as a means of sequence-directed fragmentation methods (as described above), trypsin can cleave to a smaller extent at the C-terminus of hydrophobic amino acid residues.

  In a further embodiment of this aspect of the invention, a sample comprising a mass labeled peptide internal standard precursor and one or more analyte proteins is separated after enzymatic or chemical cleavage.

  Fragmentation of heterogeneous protein populations into peptides usually results in a higher complex mixture of peptides and requires separation prior to mass spectrometry to reduce complexity. Methods that may be used for peptide separation include liquid chromatography (in one or more separation dimensions), solid phase extraction, and affinity capture, as will be understood by those skilled in the art.

Separation is either online (liquid chromatography electrospray mass spectrometry) or by fractionation followed by analysis of individual fragments, eg, by matrix-assisted laser desorption / ionization mass spectrometry (MALDI) method. Can be combined with mass spectrometry. The difference in abundance between the analyte peptide and the mass label internal standard peptide can be measured by either single stage mass spectrometry (MS) or multistage MS (MS / MS or MS ⁿ ). In two-stage mass spectrometry, a first mass separation is used to select peptides to be analyzed, then the selected peptides are fragmented and the fragments are analyzed in a second stage mass separation.

  MS / MS is a very powerful approach for analyzing complex peptide mixtures because it has the potential to separate peptides with the same molecular mass but different amino acid composition.

  The signal intensity obtained from a particular peptide by mass spectrometry depends on the concentration, molecular weight, and ionization characteristics of the peptide, and the quenching effect of other components in the sample. For example, for two peptides that are identical except for isotopic composition, the relative signal intensity will depend only on concentration, since all other factors should affect them equally (FIG. 2). ).

  A preferred method for separating peptides is “Signature Peptide Capture” (SPC), as described in PCT / EP2004 / 002566, incorporated herein by reference.

SPC is a method for analyzing heterogeneous samples of peptides or proteins, or fragments thereof, comprising the following steps:
(A) A heterogeneous sample of peptides or proteins, or fragments thereof, by binding each class of heterogeneous peptide or protein member to a spaced, defined location on the array into a heterogeneous class Separating, wherein peptides or proteins in each class have a motif common to that class; and (b) characterizing the peptides or proteins in each class;
including.

  Each heterogeneous class of peptides or proteins consists of all peptides or proteins in the heterogeneous sample that bind to specific binding molecules present on the array. Because binding molecules are selected for their ability to bind a motif rather than a particular protein or peptide, the binding molecules can bind to different types of proteins and peptides that contain the same motif. Preferably, each binding molecule is specific for a given motif. Thus, in SPC, the heterogeneous class of proteins and peptides bound by a given binding molecule typically averages at least two, more typically more than two, for example 10, Includes 20, 50, 100, 200, 500, 1000 or more different types of proteins or peptides.

  Thus, proteins and peptides are classified by SPC based on their ability to be captured and retained by specific binding molecules. A heterogeneous class of peptides or proteins will bind to a specific binding molecule due to the presence of a motif common to all members of a particular class. Thus, in each class of peptides, the identity of the motifs bound is the result of the binding specificity of the binding molecule that defines that class.

  The methods of the present invention allow SPC not only to provide analysis on heterogeneous samples of proteins or peptides, but also to quantify one or more components of the heterogeneous sample.

  A further aspect of the invention is a method of assisting in diagnosing whether a patient has a disease characterized by an increase or decrease in one or more polypeptide levels, or is likely to be susceptible to the disease. A method comprising obtaining a test sample from the patient and quantifying the one or more polypeptide levels using the method of the first aspect of the invention.

  The methods of the invention can be used in conjunction with other diagnostic methods to determine whether a patient has a disease or is likely to be afflicted with a disease. This is because the method of the present invention can be used to quantify protein or peptide levels in a test sample. Thus, using the methods of the invention can aid in the diagnosis of diseases that result in an increase or decrease in the level of one or more polypeptides.

  “Test samples” include samples of body fluids such as blood, serum, plasma, urine, cerebrospinal fluid, pleural fluid, and semen.

  Examples of diseases that can result in an increase or decrease in one or more polypeptide levels include cancer, stroke, metabolic disease, inflammation, myocardial infarction, or atherosclerosis.

  Further aspects of the invention include an N-terminal extension of at least one amino acid relative to the most N-terminal N-terminus of the mass-labeled peptide internal standard originating from the precursor and the most C-terminal side of the mass-labeled peptide internal standard originating from the precursor A mass-labeled peptide internal standard precursor comprising a C-terminal extension of at least one amino acid relative to the C-terminus of wherein the precursor is one or more upon exposure to enzymatic or chemical cleavage Generate a plurality of mass-labeled peptide internal standards.

  The term “mass label peptide internal standard precursor” includes those peptides or proteins as defined in connection with the first aspect of the invention. Thus, an internal standard precursor will produce fragments that are identical or nearly identical to fragments derived from one or more proteins to be analyzed, except for slightly different molecular weights, upon fragmentation with proteases or chemicals.

  A method of enzymatic or chemical cleavage of a mass label peptide internal standard precursor is described above in connection with the first aspect of the invention.

  The amino acid sequence of the mass label peptide internal standard precursor will necessarily vary depending on the target protein or peptide to be analyzed, as will be appreciated by those skilled in the art.

  In a further aspect of the invention, a mass-labeled peptide internal standard precursor as defined above or as defined in connection with the first aspect of the invention and an agent for fragmenting the peptide. Provide kit of parts including.

  In a further aspect of the invention, a test sample comprising a mass labeled peptide internal standard precursor as defined above or as defined in connection with the first aspect of the invention and an analyte protein or peptide, or A kit of parts is provided that includes a test sample to be tested (eg, considered to contain) the protein or peptide to be analyzed. The kit may further include an agent for fragmenting the peptide.

  As described above, the mass label peptide internal standard precursor comprises an N-terminal extension of at least one amino acid relative to the most N-terminal N-terminus of the mass label peptide internal standard resulting from the precursor, and the mass label peptide internal standard resulting from the precursor A C-terminal extension of at least one amino acid relative to the standard C-terminal C-terminus.

  Agents suitable for fragmenting peptides are described above in connection with the first aspect of the invention. The agent to be supplied as part of the kit will necessarily vary depending on the fragmentation characteristics of the mass label peptide internal standard precursor, e.g. the precursor is the correct mass label peptide internal standard after digestion with triprin. The kit will contain trypsin.

  The kit may also include additional reagents for use in the methods of the invention, such as an affinity array, antibody, or affinity column in a chip format, as will be understood by those skilled in the art.

  Preferably, the kit also includes all components required to perform SPC-mediated separation of the fragmented peptides as described above in connection with the first aspect of the invention. Good.

  Thus, the kit may also include an array having a number of different types of binding molecules, each immobilized in a separate region on a solid support (typically glass or polymer). Each binding molecule has the ability to specifically bind to a motif rather than a particular protein or peptide, as described above in connection with the first aspect of the invention.

  All documents mentioned in this specification are incorporated herein by reference in order to avoid misunderstandings.

  The invention will now be described by reference to the following non-limiting drawings and examples.

[Example]
(Example 1: Quantification of transferrin in human serum)
Transferrin is an iron-binding transport protein that is involved in the transport of iron from adsorption and heme degradation sites to storage and utilization sites. Serum transferrin may also have an additional role in stimulating cell proliferation. Transferrin plasma levels increase during pregnancy and iron deficiency and can be increased by inflammation, tumors, nephrosis, and hemochromatosis. Normal plasma levels are 2-4 mg / mL.

An internal peptide standard precursor having the amino acid sequence “EPNNK EGYYGYTGAFR * CLVE” was synthesized, where arginine is isotopically labeled by incorporating six ¹³ C atoms. This peptide is dissolved in phosphate buffered saline (PBS) at pH 7.4 at a concentration of 0.5 mg / mL.

(Sample preparation)
A 0.1 mL serum sample to be analyzed is mixed with 0.9 mL of internal peptide standard precursor solution. The sample is reduced by adding 220 μL of 75 mM dithiothreitol in PBS and incubated at 50 ° C. for 80 minutes. 60 μL of 1M iodoacetic acid in PBS is added and the sample is incubated for 30 minutes at 37 ° C. in the dark. Residual iodoacetic acid is reduced by adding 220 μL of 75 mM dithiothreitol in PBS. Reduced and alkylated protein is separated from excess reagent by gel filtration on a PD-10 column (Amersham Biotech) according to the protocol provided by the manufacturer. The protein fraction is eluted with 3 mL PBS at a protein concentration of approximately 2.5 mg / mL. 150 μL of trypsin solution (1 mg / mL trypsin in PBS) is added and the sample is incubated at 37 ° C. for 2 hours. If not analyzed immediately, the digested sample is frozen at -80 ° C.

(Peptide separation)
Peptides are separated by reverse phase high performance liquid chromatography on a capillary column (100 μm × 10 cm) using a C18 stationary phase at a flow rate of 2 μL / min. Load 10 μL of sample and elute peptide for 45 min with 5-60% phase B mobile phase gradient (eluent A: 2% acetonitrile, 0.1% trifluoroacetic acid, eluent B: 95% Acetonitrile, 0.1% trifluoroacetic acid). The eluted peptide is collected in 2 μL fractions directly on the MALDI-TOF metal target and dried.

  To identify the fractions containing the relevant peptides, 0.9 mL of internal peptide standard precursor solution is prepared as described above, replacing the serum sample with 0.1 mL of PBS. As described above, the peptides are separated and collected on another MALDI target. The MALDI target plate is scanned as below to localize the peptide on the plate.

(Mass spectrometry)
1 μL of α-cyano-sinapic acid solution (5 mg / mL in 1% trifluoroacetic acid) is added to each fraction. Fractions are analyzed using a MALDI-TOF mass spectrometer equipped with a reflector.

The transferrin-derived analyte peptide with the “EGYYGYTGAFR” sequence produced a signal corresponding to a mass of 1284.57 daltons (1283.57 plus 1 proton), while the internal peptide standard was 1280.57 daltons. Detected as having mass. The relative intensity of the signal will reflect the relative amounts of the analyte peptide and the reference peptide.
[References]

FIG. 1 shows the cleavage of the protein to be analyzed and the mass labeled peptide internal standard precursor that yields an analyzable peptide from the protein and an internal standard peptide from the internal standard precursor. FIG. 2 shows a method for analyzing a biological sample and a mass shift peptide internal standard precursor. FIG. 3 shows the mass analysis results of the analyte and the internal standard peptide.

Claims (19)

In order to generate one or more mass label peptide internal standards from one or more mass label peptide internal standard precursors, the sample to be analyzed and one or more mass label peptide internal standard precursors, Including exposing to enzymatic or chemical cleavage together;
Wherein the mass label peptide internal standard precursor comprises an N-terminal extension of at least one amino acid relative to the most N-terminal N-terminus of the mass label peptide internal standard resulting from the precursor, and a mass label peptide internal standard resulting from the precursor A C-terminal extension of at least one amino acid relative to the most C-terminal C-terminus of
A quantitative measurement method using mass spectrometry of one or more peptides produced from one or more analyte proteins by enzymatic or chemical cleavage.   The N-terminal extension is present in the analyte protein (before cleavage) in the sequence of the analyte protein corresponding to the N-terminal side of the mass label peptide internal standard generated from the precursor. The method of claim 1, comprising an amino acid sequence of at least 1, 2, 3, 4, or 5 amino acids identical to an adjacent amino acid sequence at the C-terminal end of the extension.   In the sequence of the protein to be analyzed corresponding to the most C-terminal side of the mass label peptide internal standard generated from the precursor, the C-terminal extension is present in the protein to be analyzed (before cleavage). The method of claim 1, comprising an amino acid sequence of at least one, two, three, four, or five amino acids identical to a neighboring amino acid sequence at the N-terminal end of the extension.   The amino acid sequence of at least 1, 2, 3, 4, or 5 amino acids adjacent to the N-terminus of the mass label peptide internal standard arising from the precursor corresponds to the sequence of the mass label peptide internal standard. The method according to any one of claims 1 to 3, wherein the sequence of the protein to be analyzed is identical to the amino acid sequence adjacent to the N-terminal sequence present in the protein to be analyzed (before cleavage).   Protein to be analyzed whose amino acid sequence of at least 2, 3, 4, or 5 amino acids adjacent to the C-terminus of the mass label peptide internal standard resulting from the precursor corresponds to the sequence of the mass label peptide internal standard The method according to any one of claims 1 to 4, wherein the sequence is identical to the amino acid sequence adjacent to the C-terminal sequence present in the protein to be analyzed (before cleavage).   6. The method according to any one of claims 1 to 5, wherein each mass label peptide internal standard has a length of 4 to 50 amino acids.   6. The method according to any one of claims 1 to 5, wherein each mass label peptide internal standard has a length of 10 to 40 amino acids.   The method according to any one of claims 1 to 7, wherein the mass label peptide internal standard precursor has a length of 6 to 200 amino acids.   9. The method of any one of claims 1 to 8, wherein the mass label peptide internal standard precursor comprises two or more mass label peptide internal standards.   10. The method of claim 9, wherein the mass label peptide internal standard precursor comprises a plurality of mass label peptide internal standards for analyzing peptide fragments derived from a plurality of analyte proteins.   2. The mass labeled peptide internal standard precursor is co-purified with one or more analytes to be measured during sample preparation performed prior to exposure to enzymatic or chemical cleavage. The method of any one of thru | or 10.   12. The mass label peptide internal standard precursor is combined with a sample to be analyzed after sample collection but prior to any sample preparation or fragmentation step. Method.   The method according to any one of claims 1 to 12, wherein the enzymatic or chemical cleavage is sequence-directed.   14. The method according to any one of claims 1 to 13, wherein the mass label peptide internal standard precursor and a sample comprising one or more analyte proteins are separated after enzymatic or chemical cleavage.   15. The method of claim 14, wherein the separation is performed using signature peptide capture.   A method for assisting in diagnosing whether a patient has a disease characterized by an increase or decrease in one or more polypeptide levels, or is likely to be afflicted with the disease, comprising: 16. A method comprising obtaining a test sample and quantifying the one or more polypeptide levels using a method as defined in any one of claims 1-15.   At least one N-terminal extension of at least one amino acid to the N-terminal end of the mass-labeled peptide internal standard most N-terminal, and at least one to the C-terminal end of the mass-labeled peptide internal standard most C-terminal A mass label peptide internal standard precursor that includes a C-terminal extension of an amino acid and generates one or more mass label peptide internal standards upon exposure to enzymatic or chemical cleavage.   A kit of parts comprising a mass labeled peptide internal standard precursor as defined in claim 17 or as defined in any one of claims 1 to 16 and an agent for fragmenting the peptide.   A test sample comprising a mass-labeled peptide internal standard precursor and an analyte protein or peptide as defined in claim 17 or as defined in any one of claims 1 to 16, or an analyte protein or peptide A kit of parts including a test sample to be tested for.

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