Absolute Quantification of Proteins and Modified

(19) &

(11) EP 1 472 539 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: G01N 33/53 (2006.01) 04.05.2011 Bulletin 2011/18 (86) International application number: (21) Application number: 02757101.7 PCT/US2002/025778

(22) Date of filing: 14.08.2002 (87) International publication number: WO 2003/016861 (27.02.2003 Gazette 2003/09)

(54) ABSOLUTE QUANTIFICATION OF PROTEINS AND MODIFIED FORMS THEREOF BY MULTISTAGE MASS SPECTROMETRY ABSOLUTE QUANTIFIZIERUNG VON PROTEINEN UND MODIFIZIERTEN FORMEN DAVON MITTELS MEHRSTUFIGER MASSENSPEKTROMETRIE QUANTIFICATION ABSOLUE DE PROTEINES ET DE FORMES MODIFIEES DE PROTEINE PAR SPECTROMETRIE DE MASSE MULTISTADE

(84) Designated Contracting States: • BARR J R ET AL: "Isotope dilution-mass AT BE BG CH CY CZ DE DK EE ES FI FR GB GR spectrometric quantification of specific proteins: IE IT LI LU MC NL PT SE SK TR model application with apoliprotein A-I" CLINICAL CHEMISTRY, AMERICAN (30) Priority: 14.08.2001 US 312279 P ASSOCIATION FOR CLINICAL CHEMISTRY, WASHINGTON, DC, US, vol. 42, no. 10, 1996, (43) Date of publication of application: pages 1676-1682, XP002964160 ISSN: 0009-9147 03.11.2004 Bulletin 2004/45 • SANNOLO NICOLA ET AL: "Biomonitoring of human exposure to methyl bromide by isotope (73) Proprietor: President and Fellows of Harvard dilution mass spectrometry of peptide adducts" College JOURNAL OF MASS SPECTROMETRY, vol. 34, Cambridge, MA 02138 (US) no. 10, October 1999 (1999-10), pages 1028-1032, XP009060641 ISSN: 1076-5174 (72) Inventors: • ZHOU H ET AL: "A systematic approach to the • GYGI, Steven. P. analysis of protein phosphorylation" NATURE Foxboro, MA 02035 (US) BIOTECHNOLOGY, NATURE PUBLISHING, US, • GERBER, Scott, Anthony vol. 19, April 2001 (2001-04), pages 375-378, Boston, MA 02113 (US) XP002974320 ISSN: 1087-0156 • VINALE F ET AL: "Development of a stable (74) Representative: Adam, Holger et al isotope dilution assay for an accurate Kraus & Weisert quantification of protein-bound Nepsilon-(1- Patent- und Rechtsanwälte deoxy-D-fructos-1-yl)-L-l ysine using a 13C- Thomas-Wimmer-Ring 15 labeled internal standard" JOURNAL OF 80539 München (DE) AGRICULTURAL AND FOOD CHEMISTRY, vol. 47, no. 12, December 1999 (1999-12), pages (56) References cited: 5084-5092, XP002365015 ISSN: 0021-8561 WO-A-00/11208 WO-A-02/084250 • DESIDERIO D M: "Mass spectrometric analysis US-A- 4 444 879 of neuropeptidergic systems in the human pituitary and cerebrospinal fluid" JOURNAL OF CHROMATOGRAPHY B : BIOMEDICAL APPLICATIONS, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 731, no. 1, 6 August 1999 (1999-08-06), pages 3-22, XP004177426 ISSN: 0378-4347

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 1 472 539 B1

Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 1 472 539 B1

• ARNOTT ET AL.: ’Rapid identification of • CONRADS ET AL.: ’Utility of accurate mass tags comigrating gel-isolation proteins by ion trap- for proteome-wide protein identification’ massspectrometry’ ELECTROPHORESIS vol. 19, ANALYTICAL CHEMISTRY vol. 72, 2000, pages 1998, pages 968 - 980, XP002963575 3349 - 3354, XP002963573 • JI ET AL.: ’Strategy for qualitative and • MURBY ET AL.: ’Differential degradation of a quantitative analysis in proteomics based on recombinant albumin-binding receptor in signature peptides’ JOURNAL OF escherichia coli’ EUROPEAN JOURNAL OF CHROMATOGRAPHY B vol. 745, 2000, pages 197 BIOCHEMISTRY vol. 199, 1991, pages 41 - 46, - 210, XP004215299 XP002963572 • LIN ET AL.: ’C-terminal peptide sequencing via multistage mass spectrometry’ ANALYTICAL CHEMISTRY vol. 70, 1998, pages 5162 - 5165, XP002963574

2 1 EP 1 472 539 B1 2

Description Proteins from two cells lysates can be labeled independ- ently with one or the other ICAT reagent at cysteinyl res- Government Grants idues. After mixing and proteolysing the lysates, the ICAT-labeled peptides are isolated by affinity to a biotin [0001] At least part of the work contained in this appli- 5 molecule incorporated into each ICAT reagent. ICAT- la- cation was performed under government grant HG00041 beled peptides are analyzed by LC-MS/MS where they from the National Institutes of Health, U.S. Department elute as heavy and light pairs of peptides. Quantification of Health and Human Services. The government may is performed by determining the relative expression ratio have certain rights in this invention. relating to the amount of each ICAT- labeled peptide pair 10 in the sample. Field of the Invention [0007] Identification of each ICAT-labeled peptide is performed by a second stage of mass spectrometry [0002] This invention provides a method for obtaining (MS/MS) and sequence database searching. The end absolute quantification of proteins and their modifications result is relative protein expression ratios on a large directly from cell lysates. In particular, the invention pro- 15 scale. The major drawback to this technique are 1) quan- vides the use of peptide internal standards in high per- tification is only relative; 2) specialized chemistry is re- formance liquid chromatography (HPLC) with online de- quired, and 3) database searches are hindered by the tection by multistage mass spectrometry (MSn). presence of the large ICAT reagent molecule, and 4) relative amounts of posttranslationally modified (e.g., phos- Background of the Invention 20 phorylated) proteins are transparent to analysis.

[0003] There is a need to provide novel methods for Summary the quantification of proteins and modified proteins from cell lysates. The current standard for protein detection [0008] The present invention provides a method for (quantification) is based on immunoreactive detection 25 accurate quantification of proteins. The method of the (Western analysis). However, this technique requires the invention is useful for rapid, high throughput analysis of availability of an appropriately specific antibody. In addi- proteomes. tion, many antibodies only recognize proteins in an un- [0009] In particular, the present invention provides a folded (denatured) form, cross- reactivity can be severely method for determining the presence and/or quantity of limiting, and quantification is generally relative. 30 a target polypeptide in at least one mixture of different [0004] The development of methods and instrumenta- polypeptides, comprising: tion for automated, data-dependent electrospray ionization (ESI) tandem mass spectrometry (MS/MS) in con- a) providing a mixture of different polypeptides; junction with microcapillary liquid chromatography (LC) b) adding a known quantity of a peptide internal and database searching has significantly increased the 35 standard labeled with a mass-altering label, thereby sensitivityand speed of theidentification of gel- separated generating a spiked mixture, wherein the labeled proteins. Microcapillary LC-MS/MS has been used suc- peptide internal standard comprises a subsequence cessfully for the large-scale identification of individual of the target polypeptide and wherein the labeled proteins directly from mixtures without gel electrophoretic peptide internal standard possesses a known pep- separation (Link et al., 1999; Opitek et al., 1997). How- 40 tide fragment signature diagnostic of the presence ever, while these approaches dramatically accelerate of the peptide; protein identification, quantities of the analyzed proteins c) treating the spiked mixture with a protease activity cannot be easily determined, and these methods have to generate a plurality of peptides including the la- not been shown to substantially alleviate the dynamic beled peptide internal standard and peptides corre- range problem also encountered by the 2DE/MS/MS ap- 45 sponding to the target polypeptide; proach. Therefore, low abundance proteins in complex d) fragmenting by multistage mass spectrometry the samples are also difficult to analyze by the microcapillary labeled peptide internal standard and any target pep- LC/MS/MS method without their prior enrichment. tide present in the spiked mixture comprising the [0005] There is thus a need to provide methods for the same amino acid sequence as the labeled peptide accurate comparison of protein expression levels be- 50 internal standard; tween cells in two different states, particularly for com- e) determining the ratio of labeled fragments to un- parison of low abundance proteins. labeled fragments; and [0006] Another metholology has recently been de- f) calculating from the ratio and the known quantity scribed. ICAT™ reagent technology makes use of a class of the labeled internal standard, the quantity of the of chemical reagents called isotope coded affinity tags 55 target polypeptide in the mixture; optionally (ICAT). These reagents exist in isotopically heavy and g) determining the presence and/or quantity of target light forms which are chemically identical with the excep- peptides in at least two mixtures wherein one mixture tion of eight deuterium or hydrogen atoms, respectively. is from a cell having a first cell state and the other

3 3 EP 1 472 539 B1 4

mixture is from a cell having a second cell state. way and the panel is used to determine the presence and/or quantity of the activated or inactivated form of a [0010] In a method for generating a peptide internal pathway protein. standard, the method comprises identifying a real or pre- [0016] The invention provides a method for determin- dicted peptide digestion product of a target polypeptide, 5 ing the presence and/or quantity of a target polypeptide determining the amino acid sequence of the peptide di- in at least one mixture of different polypeptides. The gestion product and synthesizing a peptide having the method comprises providing a mixture of different amino acid sequence. Thepeptide islabeled with amass- polypeptides and spiking the mixture with a known quan- altering label (e.g., by incorporating labeled amino acid tity of a peptide internal standard labeled with a mass- residues during the synthesis process) and fragmented 10 altering label. The labeled peptide internal standard com- (e.g., by multi-stage mass spectrometry). Preferably, the prises a subsequence of the target polypeptide and pos- label is a stable isotope. A peptide signature diagnostic sesses a known peptide fragment signature diagnostic of the peptide is determined, after one or more rounds of the presence of the peptide subsequence. The spiked of fragmenting, and the signature is used to identify the mixture is treated with a protease activity to generate a presence and/or quantity of a peptide of identical amino 15 plurality of peptides including the labeled peptide internal acid sequence in a sample. standard and peptides corresponding to the target [0011] Preferably, a labeled peptide is provided which polypeptide. Preferably, a chromatographic separation co-elutes with an unlabeled peptide having the same ami- step is performed to isolate the labeled peptide internal no acid sequence (i.e., a target peptide) in a chromato- standard and any target peptide present in the spiked graphic separation procedure (e.g., such as HPLC). 20 mixture which comprises the same amino acid sequence [0012] In one aspect, the mass- altering label is part of as the standard. Preferably, the internal standard and a peptide comprising a modification, and the peptide is target peptide co-elute with each other. fragmented to determine a peptide signature diagnostic [0017] The labeled peptide internal standard and tar- of such a modified peptide. The modified residue in the get peptide are fragmented using multistage mass spec- peptide internal standard comprises a phosphorylated 25 trometry and the ratio of labeled fragments to unlabeled residue, a glycosylated residue, an acetylated residue, fragments is determined. The quantity of the target a ubiquitinated residue, a ribosylated residue, or a far- polypeptide can be calculated using both the ratio and nesylated residue, or another modification found in a cel- known quantity of the labeled internal standard. The mix- lular protein. In one aspect, panels of peptide internal tures of different polypeptides can include, but are not standards are generated corresponding to (i.e., diagnos- 30 limited to, such complex mixtures as a crude fermenter tic of) different modified forms of the same protein. solution, a cell-free culture fluid, a cell or tissue extract, [0013] Peptide internal standards corresponding to dif- blood sample, a plasma sample, a lymph sample, a cell ferent peptide subsequences of a single target protein or tissue lysate; a mixture comprising at least about 100 also can be generated to provide for redundant controls different polypeptides; at least about 1000 different in a quantitative assay. In one aspect, different peptide 35 polypeptides, at least about 100, 000 different polypep- internal standards corresponding to the same target pro- tides, or a mixture comprising substantially the entire tein are generated and differentially labeled (e.g., pep- complement of proteins in a cell or tissue. In one preferred tides are labeled at multiple sites to vary the amount of aspect, the method is used to determine the presence of heavy label associated with a given peptide). and/or quantity of one or more target polypeptides direct- [0014] A panel of peptide internal standards corre-40 ly from one or more cell lysates, i.e., without separating sponding to different amino acid subsequences of a sin- proteins from other cellular components or eliminating gle protein is used to scan for mutations in that protein. other cellular components. In a further aspect, peptide internal standards corre- [0018] In one aspect, the presence and/or quantity of sponding to different variant sequences of a single amino target polypeptide in a mixture are diagnostic of a cell acid subsequence of a single protein are provided. A45 state. In another aspect, the cell state is representative match between a peptide internal standard and a target of an abnormal physiological response, for example, a peptide in a sample indicates the presence of a variant physiological response which is diagnostic of a disease. sequence in the sample. In one aspect, the multiple pep- In a further aspect, the cell stateis a state ofdifferentiation tide internal standards corresponding to variant sequenc- or represents a cell which has been exposed to a condi- es are differentially labeled. 50 tion or agent (e.g., a drug, a therapeutic agent, a potential [0015] A panel of peptide internal standards corre- toxin). In one aspect, the method is used to diagnose the sponding to amino acid subsequences of different pro- presence or risk of a disease. In another aspect, the teins in a molecular pathway is generated. Molecular method is used to identify a condition or agent which pathways, include, but are not limited to signal transduc- produces a selected cell state (e.g., to identify an agent tion pathways, cell cycle pathways, metabolic pathways, 55 which returns one or more diagnostic parameters of a blood clotting pathways, and the like. In one aspect, the cell state to normal). panel includes peptide standards which correspond to [0019] In a further aspect, the method comprises de- different modified forms of one or more proteins in a path- termining the presence and/or quantity of target peptides

4 5 EP 1 472 539 B1 6 in at least two mixtures. In another aspect, one mixture standard has a unique peptide fragmentation signature is from a cell having a first cell state and the second mix- diagnostic of the peptide. The peptide is a subsequence ture is from a cell having a second cell state. In a further of a known protein and can be used to identify the pres- aspect, the first cell is a normal cell and the second cell ence of and/or quantify the protein in sample, such as a is from a patient with a disease. In still a further aspect, 5 cell lysate. In one aspect, the peptide internal standard the first cell is exposed to a condition and/or treated with comprises a label associated with a modified amino acid an agent and the second cell is not exposed and/or treat- residue, such as a phosphorylated amino acid residue, ed. Preferably, first and second mixtures are evaluated a glycosylated amino acid residue, an acetylated amino in parallel. acid residue, a famesylated residue, a ribosylated resi- [0020] Alternatively,the two mixtures can be from iden- 10 due, and the like. In another aspect, a pair of reagents tical samples or cells. In one aspect, a labeled peptide is provided, a peptide internal standard corresponding to internal standard is provided in different known amounts a modified peptide and a peptide internal standard cor- in each mixture. In another aspect, pairs of labeled pep- responding to a peptide identical in sequence but not tide internal standards are provided each comprising modified. mass-altering labels which differ in mass, e.g., by includ- 15 [0025] In one aspect, panels of peptide internal stand- ing different amounts of a heavy isotope in each peptide. ards representing different variant forms of a single ami- [0021] In a method of determining the presence of no acid subsequence of a polypeptide are used. and/or quantity of a modification in a target polypeptide, [0026] In another aspect, panels of peptide internal the label in the internal standard is part of a peptide com- standards corresponding to different amino acid subse- prising a modified amino acid residue or to an amino acid 20 quences of single polypeptide are used. residue which is predicted to be modified in a target [0027] In a further aspect, panels of peptide internal polypeptide. In one aspect, the presence of the modifi- standards are used which correspond to different pro- cation reflects the activity of a target polypeptide and the teins in a molecular pathway (e.g., a signal transduction assay is used to detect the presence and/or quantity of pathway, a cell cycle pathway, a metabolic pathway, a an active polypeptide. The method is advantageous in 25 blood clotting pathway and the like). In still a further as- enabling detection of small quantities of polypeptide pect, peptide internal standards corresponding to differ- (e.g., about 1 part per million (ppm) or less than about ent modified forms of one or more proteins in a pathway 0.001% of total cellular protein). are used. [0022] In a method for scanning for mutations in a pro- [0028] In still a further aspect, panels of peptide inter- tein sequence panels of peptide internal standards are 30 nal standards are used which correspond to proteins di- used corresponding to different variant forms of a single agnostic of different diseases, allowing a mixture of pep- sequence or multiple peptide internal standards repre- tide internal standards to be used to test for the presence senting different amino acid subsequences of a protein. of multiple diseases in a single assay. In the first scenario, a match to a variant peptide internal [0029] Kits comprising one or more peptide internal standard in a sample indicates the presence of the variant 35 standards labeled with a stable isotope can be used. In in the sample. In the second scenario, a lack of match to one aspect, a kit comprises peptide internal standards a one peptide internal standard and matches to one or comprising different peptide subsequences from a single more other peptide internal standards indicates the pres- known protein. In another aspect, the kit comprises pep- ence of a mutation in the amino acid sequence corre- tide internal standards corresponding to different variant sponding to the mismatched peptide. 40 forms of the same amino acid subsequence of a target [0023] In a method for profiling the activity of a molec- polypeptide. In still another aspect, the kit comprises pep- ular pathway panels of peptide internal standards are tide internal standards corresponding to different known used corresponding to different pathway proteins and/or or predicted modified forms of a polypeptide. In a further to different modified forms of the proteins. The presence aspect, the kit comprises peptide internal standards cor- and/or quantity of the proteins can be used to profile the 45 responding to sets of related proteins, e.g., such as pro- function of a pathway in a particular cell. In one aspect, teins involved in a molecular pathway (a signal transduc- the pathway is one or more of a signal transduction path- tion pathway, a cell cycle, etc) and/or to different modified way, a cell cycle pathway, a metabolic pathway, a blood forms of proteins in the pathway. In still a further aspect, clotting pathway and the like. The coordinate function of a kit comprises a labeled peptide internal standard as multiple pathways can be evaluated using a plurality of 50 described above and software for performing multistage panels of standards. Similarly, the peptide internal stand- mass spectrometry. The kit may also include a means ards can be used to assay for the presence of multiple for obtaining access to a database comprising data files diseases or pathological conditions by providing a panel which include data relating to the mass spectra of frag- of peptide internal standards which comprises peptide mented peptide ions generated from peptide internal internal standards diagnostic of different diseases. 55 standards. The means for obtaining access can be pro- [0024] Reagents useful for performing the method ac- vided in the form of a URL and/or identification number cording to the invention comprise a peptide internal for accessing a database or in the form of a computer standard labeled with a stable isotope. Preferably, the program product comprising the data files. In one aspect,

5 7 EP 1 472 539 B1 8 the kit comprises a computer program product which is sequences ALELFR and LFTGHPETLEK, respec- capable of instructing a processor to perform any of the tively, from the myoglobin protein. Each peptide pro- methods described above. duces a characteristic signature ion that can be used to detect and/or quantify myoglobin in a sample of Brief Description of the Figures 5 cellular proteins. Providing both peptide internal standards together in an assay can provide an ad- [0030] The objects and features of the invention can ditional control for quantification. be better understood with reference to the following detailed description and accompanying drawings. Detailed Description 10 Figure 1 is a schematic diagram illustrating a method [0031] The invention provides a method for detecting for generating a peptide internal standard for a pro- and/or quantifying proteins in complex mixtures, such as tein or modified protein to be detected and/or quan- a cell lysate. The method can be used in high through tified. put assays to profile cellular proteomes. 15 Figure 2 illustrates characterization of peptide inter- Definitions nal standards by mass- to-charge ratio and retention time in reverse phase chromatography according to [0032] The following definitions are provided for spe- one aspect of the invention. cific terms which are used in the following written descrip- 20 tion. Figures 3A and B show characterization of a peptide [0033] As used in the specification and claims, the sin- signature by multistage mass spectrometry. Figure gular form "a", "an" and "the" include plural references 3A shows a signature obtained after a second stage unless the context clearly dictates otherwise. For exam- of mass spectrometry. Figure 3B shows a signature ple, the term "a cell" includes a plurality of cells, including obtained after performing a third stage of mass spec- 25 mixtures thereof. The term "a protein" includes a plurality trometry. of proteins. [0034] "Protein", as used herein, means any protein, Figures 4A and B illustrate steps in a method for including, but not limited to peptides, enzymes, glycopro- absolute quantitation of proteins in a complex mix- teins, hormones, receptors, antigens, antibodies, growth ture of proteins. Figure 4A shows sample processing 30 factors, etc., without limitation. Presently preferred pro- steps in which a cell lysate is spiked with a known teins include those comprised of at least 25 amino acid amount of a labeled peptide internal standard ac- residues, more preferably at least 35 amino acid residues cording to the invention. Figure 4B shows mass and still more preferably at least 50 amino acid residues. spectra of a labeled peptide internal standard and The terms "polypeptide" and "protein" are generally used the corresponding unlabeled peptide in the sample. 35 interchangeably herein to refer to a polymer of amino The ratio of labeled to unlabeled peptide provides a acid residues. means to quantify the amount of unlabeled peptide [0035] As used herein, the term "peptide" refers to a in the sample. compound of two or more subunit amino acids. The sub- units are linked by peptide bonds. Figure 5A shows a peptide internal standard suitable 40 [0036] As used herein, a "target protein" or a "target for use in detecting and/or quantitating a protein polypeptide" is a protein or polypeptide whose presence comprising the amino acid sequence GFTALK. The or amount is being determined in a protein sample. The upper panel of the Figure shows the native tryptic protein/polypeptide may be a known protein (i.e., previ- peptide. The lower portion of the Figure shows a pep- ously isolated and purified) or a putative protein (i.e., pre- tide internal standard corresponding to this peptide 45 dicted to exist on the basis of an open reading frame in which comprises a stable isotope 13( C) As can be a nucleic acid sequence). seen from the Figure, the stable isotope provides a [0037] As used herein, a "protease activity" is an ac- characteristic mass difference in the two peptides tivity which cleaves amide bonds in a protein or polypep- without altering the essential chemical structure of tide.The activitymay beimplemented byan enzyme such the peptide. Figure 5B shows a peptide internal50 as a protease or by a chemical agent, such as CNBr. standard suitable for use in detecting a phosphor- [0038] As used herein, "a protease cleavage site" is ylated form of a protein comprising the amino acid an amide bond which is broken by the action of a protease sequence GFTALK. Figure 5C shows a peptide in- activity. ternal standard suitable for use in detecting a meth- [0039] As used herein, a "labeled peptide internal ylated form of the amino acid sequence GFTALK. 55 standard" refers to a synthetic peptide which corresponds in sequence to the amino acid subsequence of Figure 6 shows diagnostic peptide fragmentation a known protein or a putative protein predicted to exist signatures obtained for two peptides comprising the on the basis of an open reading frame in a nucleic acid

6 9 EP 1 472 539 B1 10 sequence and which is labeled by a mass-altering label methods. A peptide fragmentation signature which is "di- such as a stable isotope. The boundaries of a labeled agnostic" or a "diagnostic signature" of a target protein peptide internal standard are governed by protease or target polypeptide is one which is reproducibly ob- cleavage sites in the protein (e.g., sites of protease di- served when a peptide digestion product of a target pro- gestion or sites of cleavage by a chemical agent such as 5 tein/polypeptide identical in sequence to the peptide por- CNBr). Protease cleavage sites may be predicted cleav- tion of a peptide internal standard, is fragmented and age sites (determined based on the primary amino acid which differs only from the fragmentation pattern of the sequence of a protein and/or on the presence or absence peptide internal standard by the mass of the mass- alter- of predicted protein modifications, using a software mod- ing label. Preferably, a diagnostic signature is unique to eling program) or may be empirically determined (e.g., 10 the target protein (i.e., the specificity of the assay is at by digesting a protein and sequencing peptide fragments least about 95%, at least about 99%, and preferably, ap- of the protein). In one aspect, a labeled peptide internal proaches 100%). standard includes a modified amino acid residue. [0042] A "relational" database as used herein means [0040] "Percent identity" and "similarity" between two a database in which different tables and categories of sequences can be determined using a mathematical al- 15 the database are related to one another through at least gorithm (see, e.g., Computational Molecular Biology, one common attribute and is used for organizing and Lesk, A. M., ed., Oxford University Press, New York, retrieving data. 1988; Biocomputing: Informatics and Genome Projects, [0043] The term "external database" as used herein Smith, D. W., ed., Academic Press, New York, 1993; refers to publicly available databases that are not a re- Computer Analysis of Sequence Data, Part 1, Griffin, A. 20 lational part of the internal database, such as GenBank M., and Griffin, H. G., eds., Humana Press, New Jersey, and Blocks. 1994; Sequence Analysis in Molecular Biology, von Hein- [0044] As used herein, an "expression profile" refers je, G., Academic Press, 1987; and Sequence Analysis to measurement of a plurality of cellular constituents that Primer, Gribskov, M. and Devereux, J., eds., M Stockton indicate aspects of the biological state of a cell. Such Press, New York, 1991). For example, the percent iden- 25 measurements may include, e.g., abundances or pro- tity between two amino acid sequences can be deter- teins or modified forms thereof. mined using the Needleman and Wunsch algorithm (J. [0045] As used herein, a "cell state profile" refers to Mol. Biol. (48): 444-453, 1970) which is part of the GAP values of measurements of levels of one or more proteins program in the GCG software package (available at http: in the cell. Preferably, such values are obtained by de- //www/.geg.com), by the local homology algorithm of30 termining the amount of peptides in a sample having the Smith & Waterman (Adv. Appl. Math. 2: 482, 1981), by same peptide fragmentation signatures as that of peptide the search for similarity methods of Pearson & Lipman internal standards corresponding to the one or more pro- (Proc. Natl. Acad. Sci. USA 85: 2444, 1988) and Altschul, teins. A "diagnostic profile" refers to values that are di- et al. (Nucleic Acids Res. 25(17): 3389-3402, 1997), by agnostic of a particular cell state, such that when sub- computerized implementations of these algorithms35 stantially the same values are observed in a cell, that cell (GAP, BESTFIT, FASTA, and BLAST in the Wisconsin may be determined to have the cell state. For example, Genetics Software Package (available from, Genetics in one aspect, a cell state profile comprises the value of Computer Group, 575 Science Dr., Madison, Wis.), or by a measurement of p53 expression in a cell. A diagnostic manual alignment and visual inspection (see, e.g., Au- profile would be a value which is significantly higher than subel et al., supra). Gap parameters can be modified to 40 the value determined for a normal cell and such a profile suit a user’s needs. For example, when employing the would be diagnostic of a tumor cell. A "test cell state pro- GCG software package, a NWSgapdna.CMP matrix and file" is a profile which is unknown or being verified. a gap weight of 40, 50, 60, 70, or 80 and a length weight [0046] As used herein, a processor that "receives a of 1, 2, 3, 4, 5, or 6 can be used. Examplary gap weights diagnostic profile" receives data relating to the values using a Blossom 62 matrix or a PAM250 matrix, are 16, 45 diagnosticof a particular cell state. Forexample, theproc- 14, 12,10, 8, 6, or 4, while exemplary length weights are essor may receive the values by accessing a database 1,2, 3,4, 5, or 6. The percent identity between two amino where such values are stored through a server in com- acid or nucleotide sequences also can be determined munication with the processor. using the algorithm of E. Myers and W. Miller (CABIOS 4: 11-17, 1989) which has been incorporated into the 50 Labeled Peptide Internal Standards ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap pen- [0047] Labeled peptide internal standards are used in alty of 4. determining the presence of, and/or quantifying the [0041] As used herein, "a peptide fragmentation sig- amount of, a target protein in a sample which comprises nature" refers to the distribution of mass- to-charge ratios 55 an amino acid subsequence identical to the peptide por- of fragmented peptide ions obtained from fragmenting a tion of the internal standard. Peptide internal standards peptide, for example, by collision induced disassociation, are generated by examining the primary amino acid se- ECD, LID, PSD, IRNPD, SID, and other fragmentation quence of a protein and synthesizing a peptide compris-

7 11 EP 1 472 539 B1 12 ing the same sequence as an amino acid subsequence vention, a peptide is selected from a region of a protein of the protein (see, e.g., Figure 1). In one aspect, the comprising a proline amino acid residue. peptide’s boundaries are determined by predicting the [0052] In another aspect, a peptide is selected from a cleavage sites of a protease. In another aspect, a protein region of a protein which is not expected or not known is digested by the protease and the actual sequence of 5 to be modified, so that the peptide internal standard can one or more peptide fragments is determined. Suitable be used to determine the quantity of all forms of the pro- proteases include, but are not limited to one or more of: tein. However, in a further aspect, the peptide internal serine proteases (e.g., such as trypsin, hepsin, SCCE, standard does include an amino acid residue which is TADG12, TADG14); metallo proteases (e.g., such as expected to, or is known to be modified, to provide an PUMP-1); chymotrypsin; cathepsin; pepsin; elastase; 10 internal standard to quantify only the modified form the pronase; Arg-C; Asp-N; Glu-C; Lys-C; carboxypeptidas- protein (see, e.g., Figures 5B and 5C). Peptide standards es A, B, and/or C; dispase; thermolysin; cysteine pro- representing modified (e.g., Figures 5B and 5C) and un- teases such as gingipains, and the like. Proteases may modified forms of a protein (see, e.g., Figure 5A) can be be isolated from cells or obtained through recombinant used together, to determine the extent of protein modifi- techniques. Chemical agents with a protease activity also 15 cation in a particular sample of proteins, i.e., to determine can be used (e.g., such as CNBr). what fraction of the total amount of protein is represented [0048] The target protein can be a known protein or a by the modified form. protein predicted to exist on the basis of an open reading [0053] The peptide is synthesized using one or more frame in a nucleic acid sequence. Such open reading labeled amino acids (i.e., the label is actually part of the frames can be identified from a database of sequences 20 peptide) or less preferably, labels may be attached after including, but not limited to, the GenBank database, EM- synthesis. By providing the label as part of the peptide BL data library, the Protein Sequence Database and PIR- (see, e.g., Figures 5A-5C), there are minimal differences International, SWISS-PROT, The ExPASy proteomics in the chemical structure of a peptide internal standard server of the Swiss Institute of Bioinformatics (SIB) and and a native peptide obtained from the digestion of a databases described in PCT/US01/25884. Predicted25 target protein with a protease activity. Further, because cleavage sites also can be identified through modeling the peptide is synthesized, it is unnecessary to separate software, such as IVIS-Digest (available at http://pros- and/or purify the peptide from other cellular proteins. pector.ucsf.edu/). Predicted sites of protein modification [0054] Preferably, the label is a mass-altering label. also can be determined using software packages such The type of label selected is generally based on the fol- as Scansite, Findmod, NetOGlyc (for prediction of type- 30 lowing considerations: The mass of the label should pref- O-glycosylation sequences), YinOYang (for prediction of erably be unique to shift fragment masses produced by O-beta-GlcNac attachment sites), big-PI Predictor (for MS analysis to regions of the spectrum with low back- prediction of GPI modifications), NetPhos (for prediction ground. The ion mass signature component is the portion of Ser, Thr, and Tyr phosphorylation sites), NMT (for pre- of the labeling moiety which preferably exhibits a unique diction of N-terminal N-myristolation) and Sulfinator (for 35 ion mass signature in mass spectrometric analyses. The prediction of tyrosine sulfation sites) which are accessible sum of the masses of the constituent atoms of the label through http://au.expasy.org/tools/#ptm, for example. is preferably uniquely different than the fragments of all [0049] A peptide sequence within a target protein is the possible amino acids. As a result, the labeled amino selected according to one or more criteria to optimize the acids and peptides are readily distinguished from unla- use of the peptide as an internal standard. Preferably, 40 beled amino acids and peptides by their ion/ mass pattern the size ofthe peptide isselected to minimizethe chances in the resulting mass spectrum. In a preferred embodi- that the peptide sequence will be repeated elsewhere in ment, the ion mass signature component imparts a mass other non-target proteins. Preferably, therefore, a pep- to a protein fragment produced during mass spectromet- tide is at least about 6 amino acids. The size of the peptide ric fragmentation that does not match the residue mass is also optimized to maximize ionization frequency. Thus, 45 for any of the 20 natural amino acids. peptides longer than about 20 amino acids are not pre- [0055] The label should be robust under the fragmen- ferred. In one aspect, an optimal peptide ranges from tationconditions of MS andnot undergo unfavorable frag- about 6 amino acids to about 20 amino acids, and pref- mentation. Labeling chemistry should be efficient under erably from about 7 amino acids to about 15 amino acids. a range of conditions, particularly denaturing conditions [0050] A peptide sequence is also selected which is 50 and the labeled tag preferably remains soluble in the MS not likely to be chemically reactive during mass spec- buffer system of choice. Preferably, the label does not trometry. Thus, peptide sequences which comprise suppress the ionization efficiency of the protein. More cysteine, tryptophan or methionine residues are avoided. preferably, the label does not alter the ionization efficien- [0051] Peptides also are selected based on the pres- cy of the protein and is not otherwise chemically reactive. ence of one or more bonds that preferentially fragment. 55 Alternatively, or additionally, the label contains a mixture For example, because peptides will preferentially frag- of two or more isotopically distinct species to generate a ment at proline residues, intense fragment ions may be unique mass spectrometric pattern at each labeled frag- produced at proline. Therefore in one aspect of the in- ment position.

8 13 EP 1 472 539 B1 14

[0056] In one preferred aspect, peptide internal stand- [0060] Preferably, a peptide internal standard is ana- ards comprise mass-altering labels which are stable iso- lyzed by at least two stages of mass spectrometry to de- topes. In certain preferred embodiments, the method uti- termine the fragmentation pattern of the peptide and to lizes isotopes of hydrogen, nitrogen, oxygen, carbon, or identify a peptide fragmentation signature (see, e.g., Fig- sulfur. Suitable isotopes include, but are not limited5 ure 3A). More preferably, a peptide signature is obtained to, 2H, 13C, 15N, 17O, 18O, or34S. In another aspect, in which peptide fragments have significant differences pairs of peptide internal standards can be provided, com- in m/z ratios to enable peaks corresponding to each frag- prising identical peptide portions but distinguishable la- ment to be well separated. Still more preferably, signa- bels, e.g., peptides may be labeled at multiple sites to tures are unique, i.e., diagnostic of a peptide being iden- provide different heavy forms of the peptide). Multiple 10 tified and comprising minimal overlap with fragmentation labeled amino acids may be incorporated in a peptide patterns of peptides with different amino acid sequences. during the synthesis process. In another aspect, the label If a suitable fragment signature is not obtained at the first is part of a peptide comprising a modified amino acid stage, additional stages of mass spectrometry are per- residue, such as a phosphorylated residue (see, e.g., formed until a unique signature is obtained (see, e.g., Figure 5B), a glycosylated residue, an acetylated resi- 15 Figure 3B). due, a ribosylated residue, or a famesylated residue, a [0061] Fragment ions in the MS/MS and MS3 spectra methlyated residue (see, e.g., Figure 5C). In this embod- are generally highly specific and diagnostic for peptides iment, pairs or larger sets of peptide internal standards of interest. In contrast, to prior art methods, the identifi- corresponding to modified and unmodified peptides also cation of peptide diagnostic signatures provides for a way can be produced. In one aspect, such a pair/set is differ- 20 to perform highly selective analysis of a complex protein entially labeled. mixture, such as a cellular lysate in which there may be [0057] Peptide internal standards are characterized greater than about 100, about 1000, about 10,000, or according to their mass- to-charge ratio (m/z) and prefer- even about 100,000 different kinds of proteins. Thus, ably, also according to their retention time on a chroma- while conventional mass spectroscopy would not be able tographic column (e.g., such as an HPLC column). Inter- 25 to distinguish between peptides with different sequences nal standards are selected which co-elute with peptides but similar m/z ratios (which would tend to co-elute with of identical sequence but which are not labeled (see, e.g., any labeled standard being analyzed), the use of peptide Figure 2). fragmentation methods and multistage mass spectrom- [0058] The peptide internal standard is then analyzed etry in conjunction with LC methods, provide a way to by fragmenting the peptide. Fragmentation can 30 be detect and quantitate target proteins which are only a achieved by inducing ion/molecule collisions by a proc- small fraction of a complex mixture (e.g., present in less ess known as collision-induced dissociation (CID) (also than 2000 copies per cell or less than about 0.001 % of known as collision-activated dissociation (CAD)). Colli- total cellular protein) through these diagnostic signa- sion-induced dissociation is accomplished by selecting tures. a peptide ion of interest with a mass analyzer and intro- 35 [0062] Multiple peptide subsequences of a single pro- ducing that ion into a collision cell. The selected ion then tein may be synthesized, labeled, and fragmented to collides with a collision gas (typically argon or helium) identify optimal fragmentation signatures. However, in resulting in fragmentation. Generally, any method that is one aspect at least two different peptides are used as capable of fragmenting a peptide is encompassed within internal standards to identify/quantify a single protein, the scope of the present invention. In addition to CID, 40 providing an internal redundancy to any quantitation sys- other fragmentation methods include, but are not limited tem (see, e.g., as shown in Figure 6). In another aspect, to, surface induced dissociation (SID) (James and peptide internal standards are synthesized which corre- Wilkins, Anal. Chem. 62: 1295-1299, 1990; and Williams, spond to a single amino acid subsequence of a target et al., J Amer. Soc. Mass Spectrom. 1: 413-416, 1990), polypeptide but which vary in one or more amino acids. blackbody infrared radiative dissociation (BIRD); elec- 45 The peptide internal standards may correspond to known tron capture dissociation (ECD) (Zubarev, et al., J. Am. variants or mutations in the target polypeptide or can be Chem. Soc. 120: 3265- 3266, 1998); post- source decay randomly varied to identify all possible mutations in an (PSD), LID, and the like. amino acid sequence. [0059] The fragments are then analyzed to obtain a [0063] In one preferred aspect, peptide internal stand- fragment ion spectrum. One suitable way to do this is by 50 ards corresponding to proteins expressed from nucleic CID in multistage mass spectrometry (MS n). Traditionally acids comprising single nucleotide polymorphisms are used to characterize the structure of a peptide and/or to synthesized to identify variant proteins encoded by such obtain sequence information, it is a discovery of the nucleic acids. Thus, peptide internal standards can be present invention, that MS n provides enhanced sensitiv- generated corresponding to SNP’s which map to coding ity in methods for quantitating absolute amounts of pro- 55 regions of genes and can be used to identify and quantify teins. Thus, in one aspect, peptide internal standards are variant protein sequences on an individual or population generated for low abundance proteins (e.g., below 2000 level. SNP sequences can be accessed through The Hu- copies/cell). man SNP database available at

9 15 EP 1 472 539 B1 16 http: //www- genome.wi.mit.edu/SNP/ human/ in- prises substantially all of the proteins in a cell. In one dex.html. preferred aspect, as shown in Figure 4A, a complex mix- [0064] Peptide internal standards may also be used to ture of cellular proteins is evaluated directly from a cell scan for mutations in proteins including, but not limited lysate, i.e., without any steps to separate and/or purify to, BRCA1, BRCA2, CFTR, p53, blood group antigens, 5 and/or eliminate cellular components or cellular debris. HLA proteins, MHC proteins, G- Protein Coupled Recep- [0068] While the methods described herein are com- tors, apolipoprotein E, kinases (e.g., such as hCds1, patible with any biochemical, immunological or cell bio- MTKs, PTK, CDKs, STKs, CaMs, and the like) (see, e.g., logical fractionation methods that reduce sample com- United States Patent No. 6,426,206), phosphatases, hu- plexity and enrich for proteins of low abundance, it is a man drug metabolizing proteins, viral proteins such as a 10 particular advantage of the method that it can be used viral envelope proteins (e.g., HIV envelope proteins), to detect and quantitate peptides in complex mixtures of transporter proteins, and the like. polypeptides, such as cell lysates. Unlike methods in the [0065] In a further aspect, peptides corresponding to prior art, because the present invention detects diagnos- different modified forms of a protein are synthesized, pro- tic signatures that are highly selective for individual pep- viding internal standards to detect and/or quantitate15 tides, the quantities of such peptides can be discerned changes in protein modifications in different cell states. even in a mixture of peptides of similar mass/charge ra- In still a further aspect, peptide internal standards are tios. generated which correspond to different proteins in a mo- [0069] Generally, the sample will have at least about lecular pathway and/or modified forms of such proteins 0.01 mg of protein, at least about 0.05 mg, and usually (e.g., proteins in a signal transduction pathway,cell cycle, 20 at least about 1 mg of protein or 10 mg of protein or more, metabolic pathway, blood clotting pathway, etc.) provid- typically at a concentration in the range of about 0.1-10 ing panels of internal standards to evaluate the regulated mg/ml. The sample may be adjusted to the appropriate expression of proteins and/or the activity of proteins in a buffer concentration and pH, if desired. particular pathway. Combinations of the above-de- [0070] In one aspect, as shown in Figure 4A, a known scribed internal standards can be used in a given assay. 25 amount of a labeled peptide internal standard corresponding to a target protein to be detected and/or quan- Methods of Using Peptide Internal Standards titated, is added to a sample such as a cell lysate. Pref- erably, about 10 femtomoles is spiked into the sample. [0066] The labeled peptide internal standards can be The sample is contacted with a protease activity (e.g., used to facilitate quantitative determination of the relative 30 one or more proteases or appropriate chemical agent (s) amounts of proteins in different samples. Also, the use are added to the sample) and the spiked sample is incu- of differentially isotopically labeled reagents as internal bated for a suitable period of time to allow peptide diges- standards facilitates quantitative determination of the ab- tion. If the target protein is present in the sample, the solute amounts of one or moreproteins present in asingle digestion step should liberate a target peptide identical sample. Samples that can be analyzed by method of the 35 in sequence to the peptide portion of the internal standard invention include, but are not limited to, cell homoge- and the amount of target peptides so liberated from target nates; cell fractions; biological fluids, including, but not proteins in the sample should be proportional to the limited to urine, blood, and cerebrospinal fluid; tissue ho- amount of target protein in the sample. mogenates; tears; feces; saliva; lavage fluids such as [0071] Preferably, a separation procedure is per- lung or peritoneal lavages; and generally, any mixture of 40 formed to separate a labeled peptide internal standard biomolecules, e.g., such as mixtures including proteins and corresponding target peptide from other peptides in and one or more of lipids, carbohydrates, and nucleic the sample. Representative examples include high- pres- acids such as obtained partial or complete fractionation sure liquid chromatography (HPLC), Reverse Phase- of cell or tissue homogenates. High Pressure Liquid Chromatography (RP- HPLC), elec- [0067] Preferably, a proteome is analyzed. By a pro- 45 trophoresis (e.g., capillary electrophoresis), anion or cat- teome is intended at least about 20% of total protein com- ion exchange chromatography, and open-column chro- ing from a biological sample source, usually at least about matography. Preferred is microcapillary liquid chroma- 40%, more usually at least about 75%, and generally tography. As discussed above, internal standards are se- 90% or more, up to and including all of the protein ob- lected so that they co- elute with their corresponding tar- tainable from the source. Thus, the proteome may be 50 get peptides as pairs of peptides that differ only in the present in an intact cell, a lysate, a microsomal fraction, mass contributed by the mass-altering label. an organelle, a partially extracted lysate, biological fluid, [0072] Each peptide then is examined by monitoring and the like. The proteome will be a mixture of proteins, of a selected reaction in the mass spectrometer. This generally having at least about 20 different proteins, usu- involves using the prior knowledge gained by the char- ally at least about 50 different proteins and in most cases, 55 acterization of the peptide internal standard and then re- about 100 different proteins, about 1000 different pro- quiring the mass spectrometer to continuously monitor a teins, about 10,000 different proteins, about 100,000 dif- specific ion in the MS/MS or MSn spectrum for both the ferent proteins, or more. In one aspect, a proteome com- peptide of interest and the internal standard. After elution,

10 17 EP 1 472 539 B1 18 the areas-under-the-curve (AUC) for both the peptide in- resenting different variant forms of the protein are added ternal standard and target peptide peaks are calculated to a sample as a means of detecting and/or quantifying (see, e.g., Figure 4B). The ratio of the two areas provides a particular variant form of the protein. the absolute quantification that can be normalized for the [0078] In still another aspect, peptide internal stand- number of cells used in the analysis and the protein’s 5 ards are added to a sample that represents different pro- molecular weight, to provide the precise number of cop- teins in a molecular pathway (e.g., a signal transduction ies of the protein per cell. pathway, a cell cycle, a metabolic pathway, a blood clot- [0073] In one aspect, the presence and/or quantity of ting pathway) and/or different modified forms of such pro- target polypeptide in a mixture is diagnostic of a cell state. teins. In this aspect, the function of the pathway is eval- In another aspect, the cell state is representative of an 10 uated by monitoring the presence, absence or quantity abnormal physiological response, for example, a physi- of particular pathway proteins and/or their modified ological response which is diagnostic of a disease. In a forms. Multiple pathways may be evaluated at a time by further aspect, the cell state is a state of differentiation combining mixtures of different pathway peptide internal or represents a cell which has been exposed to a condi- standards. tion or agent (e.g., a drug, a therapeutic agent, a potential 15 [0079] In a further aspect, peptide internal standards toxin). Preferably, protein quantities identified are com- represent proteins and/or modified forms thereof whose pared to a reference quantity obtained from a reference presence is diagnostic of a particular tissue type (e.g., sample (e.g., a sample from a normal patient, a sample neural proteins, cardiac proteins, skin proteins, lung pro- not exposed to a condition or agent, etc.). teins, liver proteins, pancreatic proteins, kidney proteins, [0074] In another aspect, the method comprises de- 20 proteins characteristic of reproductive organs, etc.). termining the presence and/or quantity of target peptides These can be used separately or in combination to per- in at least two mixtures. In still another aspect, one mix- form tissue-typing analysis. ture is from a cell having a first cell state and the second [0080] Peptide internal standards may represent pro- mixture is from a cell having a second cell state. In a teins or modified forms thereof whose presence is char- further aspect, the first cell is a normal cell and the second 25 acteristic of a particular genotype (e.g., such as HLA pro- cell is from a patient with a disease. Preferably, first and teins, blood group proteins, proteins characteristic of a second mixtures are evaluated in parallel. particular pedigree, etc.). These can be used separately [0075] Alternatively,the two mixtures can be from iden- or in combination to perform forensic analyses, for ex- tical samples or cells. In one aspect, the labeled peptide ample. internal standard is provided in different known amounts 30 [0081] In one aspect, peptide internal standards are in each mixture. In another aspect, pairs of labeled pep- used in prenatal testing to detect the presence of a con- tide internal standards are provided each comprising genital disease or to quantitate protein levels diagnostic mass-altering labels that differ in mass. For example, dif- of a chromosomal abnormality. ferentially labeled peptides may be generated by incor- [0082] Peptide internal standards may represent pro- porating different amounts of a heavy label into each pep- 35 teins or modified forms thereof whose presence is char- tide varying the number of sites within the peptides la- acteristic of particular diseases. Such peptides may cor- beled by a heavy isotope. respond to target proteins diagnostic of neurological dis- [0076] The invention provides a method of determining ease (e.g., neurodegenerative diseases, including, but the presence of and/or quantity of a modification in a not limited to, Alzheimer’s disease; amyotrophic lateral target polypeptide. Preferably, the label in the internal 40 sclerosis; dementia, depression; Down’s syndrome; standard is attached to a peptide comprising a modified Huntington’s disease; peripheral neuropathy; multiple amino acid residue or to an amino acid residue that is sclerosis; neurofibromatosis; Parkinson’s disease; and predicted to be modified in a target polypeptide. In one schizophrenia). These standards can be used separately aspect, multiple internal standards representing different or in combination to diagnose a neurological disease. modified forms of a single protein and/or peptides repre- 45 [0083] Preferably, sets of internal standards are used senting different modified regions of the protein are add- so that diagnostic fragmentation signatures can be eval- ed to a sample and corresponding target peptides (bear- uated for a number of different diseases in a single assay. ing the same modifications) are detected and/or quanti- Thus, a sample may be obtained from a patient who fied. Preferably, standards representing both modified presents with general symptoms associated with a neu- and unmodified forms of a protein are provided in order 50 rological disease, and a peptide internal standard mixture to compare the amount of modified protein observed to comprising internal standards for proteins diagnostic of the total amount of protein in a sample. different neurological diseases can be added to the sam- [0077] In another aspect, peptide internal standards ple. The sample is contacted with a protease activity and comprising different peptides from a single protein are peptide fractions are obtained, e.g., such as by HPLC. added in known amounts to a sample to provide addi- 55 Peptide ions are subsequently fragmented as described tional controls or to scan for mutations in different regions above to detect any diagnostic fragmentation signatures of a protein. In a further aspect, peptides corresponding present characteristic of a particular disease. The to a single amino acid subsequence in a protein but rep- uniqueness of the fragmentation signature thus allows a

11 19 EP 1 472 539 B1 20 specific diagnosis to be obtained while testing for a plu- but are not limited to: drugs; toxins; proteins; polypep- rality of different types of diseases. The peptide internal tides; peptides; amino acids; antigens; cells, cell nuclei, standard mixture may include a peptide internal standard organelles, portions of cell membranes; viruses; recep- corresponding to a control target protein, such as a con- tors; modulators of receptors (e.g., agonists, antagonists, stitutively expressed protein of known abundance. A neg- 5 and the like); enzymes; enzyme modulators (e.g., such ative standard (e.g., such as a peptide internal standard as inhibitors, cofactors, and the like); enzyme substrates; corresponding to a plant protein) may also be provided. hormones; nucleic acids (e.g., such as oligonucleotides; [0084] Similarly, peptide internal standards can be polynucleotides; genes, cDNAs; RNA; antisense mole- used to diagnose an immune disease, including, but not cules, ribozymes, aptamers), and combinations thereof. limited to, acquired immunodeficiency syndrome (AIDS); 10 Compounds also can be obtained from synthetic libraries Addison’s disease; adult respiratory distress syndrome; from drug companies and other commercially available allergies; ankylosing spondylitis; amyloidosis; anemia; sources known in the art (e.g., including, but not limited, asthma; atherosclerosis; autoimmune hemolytic anemia; to the LeadQuest® library) or can be generated through autoimmune thyroiditis; bronchitis; cholecystitis; contact combinatorial synthesis using methods well known in the dermatitis; Crohn’s disease; atopic dermatitis; dermato- 15 art. In one aspect, a compound is identified as a modu- myositis; diabetes mellitus; emphysema; episodic lym- lating agent if it alters the site of modification of a polypep- phopenia with lymphocytotoxins; erythroblastosis fetalis; tide and/or if it alters the amount of modification by an erythema nodosum; atrophic gastritis; glomerulonephri- amount that is significantly different from the amount ob- tis; Goodpasture’s syndrome; gout; Graves’ disease; served in a control cell (e.g., not treated with compound) Hashimoto’s thyroiditis; hypereosinophilia; irritable bow- 20 (setting p values to < 0.05). In another aspect, a com- el syndrome; myasthenia gravis; myocardial or pericar- pound is identified as a modulating agent, if it alters the dial inflammation; osteoarthritis; osteoporosis; pancrea- amount of the polypeptide (whether modified or not). titis; and polymyositis. [0090] Compounds identified as modulating agents [0085] Similarly, peptide internal standards can be are used in methods of treatment of pathologies associ- used to characterize infectious diseases, respiratory dis- 25 ated with abnormal sites/levels of modification or abnor- eases, reproductive diseases, gastrointestinal diseases, mal levels or types of protein. For administration to a dermatological diseases, hematological diseases, cardi- patient, one or more such compounds are generally for- ovascular diseases, endocrine diseases, urological dis- mulated as a pharmaceutical composition. Preferably, a eases, and the like. pharmaceutical composition is a sterile aqueous or non- [0086] Because peptide internal standards provide di- 30 aqueous solution, suspension or emulsion, which addi- agnostic fragmentation signatures for detecting and/or tionally comprises a physiologically acceptable carrier quantitating proteins or modified forms thereof, changes (i.e., a non-toxic material that does not interfere with the in the presence or amounts of such fragmentation sig- activity of the active ingredient). More preferably, the natures in a sample of proteins from a cell (e.g., such as composition also is non-pyrogenic and free of viruses or a cell lystate), as discussed above, can be diagnostic of 35 other microorganisms. Any suitable carrier known to a cell state. In one aspect, a single fragmentation signa- those of ordinary skill in the art may be used. Represent- ture from a peptide internal standard is diagnostic. In oth- ative carriers include, but are not limited to: physiological er aspects, sets of fragmentation signatures are diagnos- saline solutions, gelatin, water, alcohols, natural or syn- tic and multiple peptide internal standards are spiked into thetic oils, saccharide solutions, glycols, injectable or- a sample to evaluate changes in cell state. 40 ganic esters such as ethyl oleate or a combination of [0087] In one preferred embodiment, changes in cell such materials. Optionally, a pharmaceutical composi- state are evaluated after exposure of the cell to a com- tion may additionally contain preservatives and/or other pound. Compounds are selected which are capable of additivessuch as, for example, antimicrobial agents, anti- normalizing a cell state, e.g., by selecting for compounds oxidants, chelating agents and/or inert gases, and/or oth- which alter fragmentation signatures from those charac- 45 er active ingredients. teristic of abnormal physiological responses to those rep- [0091] Routes and frequency of administration, as well resentative of a normal cell. doses, will vary from patient to patient In general, the [0088] For example, a three way comparison of pharmaceutical compositions is administered intrave- healthy, diseased, and treated diseased individuals can nously, intraperitoneally, intramuscularly, subcutane- identify which compounds are able to restore a disease 50 ously, intracavity or transdermally. Between 1 and 6 dos- cell state to a one that more closely resembles a normal es is administered daily. A suitable dose is an amount cell state. This can be used to screen for drugs or other that is sufficient to show improvement in the symptoms therapeutic agents, to monitor the efficacy of treatment, of a patient afflicted with a disease associated an aber- and to detect or predict the occurrence of side effects, rant modification state or an abnormal level or type of a whether in a clinical trial or in routine treatment, and to 55 protein. Such improvement may be detected by monitor- identify protein targets which are more important to the ing appropriate clinical or biochemical endpoints as is manifestation and treatment of a disease. known in the art. In general, the amount of a modulating [0089] Compounds which can be evaluated include, agent present in a dose, or producedin situ by DNA

12 21 EP 1 472 539 B1 22 present in a dose (e.g., where the modulating agent is a used. In one preferred aspect, the database comprises polypeptide or peptide encoded by the DNA), ranges peptide diagnostic signatures, e.g., mass spectral data from about 1 Pg to about 100 mg per kg of host. Suitable obtained after fragmentation of one or more peptide in- dose sizes will vary with the size of the patient, but will ternal standards, which can be used to identify a cell hav- typically range from about 10 mL to about 500 mL for 5 ing a particular cell state. More preferably, the database 10-60 kg animal. A patient can be a mammal, such as a includes data relating to a plurality of cell state profiles, human, or a domestic animal. i.e., data relating to levels of target proteins identified by the peptide internal standards in a plurality of cells having Computer Systems and Databases different cell states. For example, profiles of disease 10 states may be included in the database and these profiles [0092] Methods for generating a database comprising will include measurements of levels of one or more pro- data files for storing information relating to diagnostic teins, or modified forms thereof, characteristic of the dis- fragmentation signatures for peptide internal standards ease state. Profiles of cells exposed to different com- are used. Preferably, data in the data files include one pounds include measurements of levels of proteins or or more peptide fragmentation signatures characteristic 15 modified forms thereof characteristic of the response(s) or diagnostic of a cell state (e.g., such as a state which of the cells to the compounds. In one aspect, the meas- is characteristic of a disease, a normal physiological re- urements are obtained by performing any of the methods sponse, a developmental process, exposure to a thera- described above. peutic agent, exposure to a toxic agent or a potentially [0097] Preferably, the database is in electronic form toxic agent, and/or exposure to a condition). Data in the 20 and the cell state profiles, which are also in electronic data files also preferably includes values corresponding form, provide measurements of levels of a plurality of to level of proteins corresponding to the peptide fragmen- proteins in a cell or cells of one or more subjects. In one tation signatures found in a particular cell state. aspect, the database comprises measurements of more [0093] In one aspect, for a cell state determined by the than about 5, more than about 10, more than about 30, differential expression of at least one protein, a data file 25 more than about 50, more than about 100, more than corresponding to the cell state will minimally comprise about 500, more than about 1000, more than about data relating to the mass spectra observed after peptide 10,000, or more than about 100,000 proteins in a cell, fragmentation of a peptide internal standard diagnostic i.e., the database comprises data relating to the pro- of the protein. Preferably, the data file will include a value teome of a cell. The measurements represent levels of corresponding to the level of the protein in a cell having 30 modified and/or unmodified forms of the proteins. In one the cell state. For example, a tumor cell state is associ- aspect, the measurements also include data regarding ated with the overexpression of p53 (see, e.g., Kern, et the site of protein modifications in one or more proteins al., Int. J. Oncol. 21(2): 243-9, 2001). The data file will in a cell. comprise mass spectral data observed after fragmenta- [0098] In one preferred aspect, cell state profiles com- tion of a labeled peptide internal standard corresponding 35 prise quantitative data relating to target proteins and/or to a subsequence of p53. Preferably, the data file also modified forms thereof obtained by using one or more of comprises a value relating to the level of p53 in a tumor the methods described above. cell. The value may be expressed as a relative value [0099] A variety of data storage structures are availa- (e.g., a ratio of the level of p53 in the tumor cell to the ble for creating a computer readable medium or memory level of p53 in a normal cell) or as an absolute value (e.g., 40 comprising data files of the database. The choice of the expressed in nM or as a % of total cellular proteins). data storage structure will generally be based on the [0094] Preferably, the data files also include informa- means chosen to access the stored information. For ex- tion relating to the presence or amount of a modified form ample, the data can be stored in a word processing text of a target a polypeptide in at least one cell and to mass file, formatted in commercially-available software such spectral data diagnostic of the modified form (i.e., peak 45 as WordPerfect and Microsoft Word, or represented in data for a fragmented peptide internal standard which the form of an ASCII file, stored in a database application, corresponds to the modified form). More preferably, the such as DB2, Sybase, Oracle, or the like. The skilled data files also comprise spectral data diagnostic of the artisan can readily adapt any number of data processor unmodified form as well as data corresponding to the structuring formats (e.g., text files, pdf files, or database level of the unmodified form. 50 structures) in order to obtain computer readable medium [0095] In one aspect, the database also comprises da- or a memory having recorded thereon data relating to ta relating to the source of a cell whose cell state is being diagnostic fragmentation signatures, e.g., such as mass evaluated. For example, the database comprises data spectral data obtained after fragmentation of the peptide relating to identifying characteristics of a patient from internal standards, and protein levels. whom the cell is derived. 55 [0100] Correlations between a particular diagnostic [0096] Further, a computer memory comprising data signature observed and a cell state (e.g., a disease, gen- files for storing information relating to the diagnostic frag- otype, tissue type, etc.) may be known or may be iden- mentation signatures of peptide internal standards is tified using the database described above and suitable

13 23 EP 1 472 539 B1 24 statistical programs, expert systems, and/or data mining user interface); a programmable central processing unit; systems, as are known in the art. and a data storage means comprising the data files and [0101] In another aspect, a computer system is used information described above, electronically stored in a comprising: a database having data files containing in- relational database. formation identifying diagnostic fragmentation signa-5 [0106] Preferably, the central processing unit compris- tures (e.g., mass spectral peaks) as corresponding to es an operating system for managing a computer and its particular peptide internal standards which in turn are network interconnections. This operating system can be, identified as corresponding to particular target proteins. for example, of the Microsoft Windows’ family, such as Preferably, the data files also comprise information for Windows 95, Windows 98, or Windows NT, or any new relating the diagnostic fragmentation signatures so iden- 10 Windows programmed developed. A software compo- tified to one or more cell states, e.g., where the target nent representing common languages may be provided. protein corresponding to the peptide internal standard is Preferred languages include C/C++, and JAVA®. In one diagnostic of a cell state, the peptide internal standard aspect, methods of thisinvention are programmedin soft- and fragmentation signature are also identified within the ware packages which allow symbolic entry of equations, data file as being diagnostic of a cell state. In one pre- 15 high-level specification of processing, and statistical ferred aspect, the system further comprises a user inter- evaluations. face allowing a user to selectively view information relating to a diagnostic fragmentation signature and to obtain Reagents and Kits information about a cell state. The interface may comprise links allowing a user to access different portions of 20 [0107] In one aspect, a reagent useful for performing the database by selecting the links (e.g. by moving a the method of the invention comprises a peptide internal cursor to the link and clicking a mouse or by using a standard labeled with a stable isotope. Preferably, the keystroke on a keypad). The interface may additionally standard has a unique peptide fragmentation signature display fields for entering information relating to a sample diagnostic of the peptide. The peptide is a subsequence being evaluated. 25 of a known protein and can be used to identify the pres- [0102] Still more preferably, the system is capable of ence of and/or quantify the protein in sample, such as a comparing diagnostic fragmentation signatures of known cell lysate. peptide internal standards to mass spectral data obtained [0108] Kits comprising one or more peptide internal for peptides in a sample spiked with one or more internal standards labeled with a stable isotope or reagents suit- standards in order to determine and/or quantify levels of 30 able for performing such labeling can be used. In certain target proteins corresponding to the standards in the preferred embodiments, the method utilizes isotopes of sample. When a match is identified, the system may also hydrogen, nitrogen, oxygen, carbon, or sulfur. Suitable provide information regarding the cell state for which the isotopes include, but are not limited 2 13 15 17 18 34 peptide internal standard is diagnostic (i.e., the system to, H, C, N, O , O or S. In another aspect, will identify the source of the cell, the compound to which 35 pairs of peptide internal standards are provided, com- a cell has been exposed, and/or a disease which the cell prising identical peptide portions but distinguishable la- is responding to). In some aspects, sets of peptide inter- bels, e.g., peptides may be labeled at multiple sites to nal standards are evaluated, as only the set will be diag- provide different heavy forms of the peptide. Pairs of pep- nostic. tide internal standards corresponding to modified and un- [0103] The system may also be used to collect and 40 modified peptides also can be provided. categorize peptide fragmentation signatures for different [0109] In one aspect, a kit comprises peptide internal types of cell states to identify sets of peptide internal standards comprising different peptide subsequences standards characteristic of particular cell states. In this from a single known protein. In another aspect, the kit aspect, preferably, the system comprises a relational da- comprises peptide internal standards corresponding to tabase. More preferably, the system further comprises 45 different known or predicted modified forms of a polypep- an expert system for identifying sets of peptide internal tide. In a further aspect, the kit comprises peptide internal standards that are diagnostic of different cell states. In standards corresponding to sets of related proteins, e.g., one aspect, the system is capable of clustering related such as proteins involved in a molecular pathway (a sig- information. Suitable clustering programs are known in nal transduction pathway, a cell cycle, etc), or which are the art and are described in, for example, U.S. Patent 50 diagnostic of particular disease states, developmental No. 6,303,297. stages, tissue types, genotypes, etc. Peptide internal [0104] The system preferably comprises a means for standards corresponding to a set may be provided in sep- linking a database comprising data files of diagnostic arate containers or as a mixture or "cocktail" of peptide fragmentation signatures to other databases, e.g., such internal standards. as genomic databases, pharmacological databases, pa- 55 [0110] In one aspect, a plurality of peptide internal tient databases, proteomic databases, and the like. standards representing a MAPK signal transduction [0105] Preferably, the system comprises in combina- pathway is used. Preferably, the kit comprises at least tion, a data entry means, a display means (e.g., graphic two, at least about 5, at least about 10 or more, of peptide

14 25 EP 1 472 539 B1 26 internal standards corresponding to any of MAPK, GRB2, a modified peptide and a peptide internal standard cor- mSOS, ras, raf, MEK, p85, KHS1, GCK1, HPK1, MEKK responding to a peptide, identical in sequence but not 1-5, ELK1, c-JUN, ATF-2, 3APK, MLK1-4, PAK, MKK, modified. p38, a SAPK subunit, hsp27, and one or more inflamma- [0118] In another aspect, one or more control peptide tory cytokines. 5 internal standards are used. For example, a positive con- [0111] In another aspect, a set ofpeptide internal trol may be a peptide internal standard corresponding to standards is used which comprises at least about two, a constitutively expressed protein, while a negative pep- at least about 5 or more, of peptide internal standards tide internal standard may be provided corresponding to which correspond to proteins selected from the group a protein known not to be expressed in a particular cell including, but not limited to, PLC isoenzymes, phosphati- 10 or species being evaluated. For example, in a kit com- dylinositol 3-kinase (PI-3 kinase), an actin-binding pro- prising peptide internal standards for evaluating a cell tein, a phospholipase D isoform, (PLD), and receptor and state in a human being, a plant peptide internal standard nonreceptor PTKs. may be provided. [0112] In another aspect, a set of peptide internal [0119] In still another aspect, a kit comprises a labeled standards is used which comprises at least about 2, at 15 peptide internal standard as described above and soft- least about 5, or more, of peptide internal standards ware for analyzing mass spectra (e.g., such as SE- which correspond to proteins involved in a JAK signaling QUEST). pathway, e.g., such as one or more of JAK 1-3, a STAT [0120] Preferably, the kit also comprises a means for protein, IL-2, TYK2, CD4, IL-4, CD45, a type I interferon providing access to a computer memory comprising data (IFN) receptor complex protein, an IFN subunit, and the 20 files storing information relating to the diagnostic frag- like. mentation signatures of one or more peptide internal [0113] In a further aspect, a set of peptide internal standards. Access may be in the form of a computer read- standards is used which comprises at least about 2, at able program product comprising the memory, or in the least about 5, or more of peptide internal standards which form of a URL and/or password for accessing an internet correspond to cytokines. Preferably, such a set compris- 25 site for connecting a user to such a memory. In another es standards selected from the group including, but not aspect, the kit comprises diagnostic fragmentation sig- limited to, pro-and anti-inflammatory cytokines (which natures (e.g., such as mass spectral data) in electronic may each comprise their own set or which may be pro- or written form, and/or comprises data, in electronic or vided as a mixed set of peptide internal standards). written form, relating to amounts of target proteins char- [0114] In still another aspect, a set of peptide internal 30 acteristic of one or more different cell states and corre- standards is used which comprises a peptide diagnostic sponding to peptides which produce the fragmentation of a cellular differentiation antigen or CD. Such kits are signatures. useful for tissue typing. [0121] The kit may further comprise expression anal- [0115] In one aspect, peptides corresponding to ysis software on computer readable medium, which is known variants or mutations in a target polypeptide, or 35 capable of being encoded in a memory of a computer which are randomly varied to identify all possible muta- having a processor and capable of causing the processor tions in an amino acid sequence, are used in the kit. In to perform a method comprising: determining a test cell a preferred aspect, peptide internal standards corre- state profile from peptide fragmentation patterns in a test sponding to proteins expressed from nucleic acids com- sample comprising a cell with an unknown cell state or prising single nucleotide polymorphisms are used. 40 a cell state being verified; receiving a diagnostic profile [0116] Peptide internal standards may include pep- characteristic of a known cell state; and comparing the tides corresponding to variant proteins selected from the test cell state profile with the diagnostic profile. group consisting of BRCA1; BRCA2; CFTR; p53; a JAK [0122] In one aspect, the test cell state profile compris- protein; a STAT protein; blood group antigens; HLA pro- es values of levels of peptides in a test sample that cor- teins; MHC proteins; G- Protein Coupled Receptors; apol- 45 respond to one or more peptide internal standards pro- ipoprotein E; kinases (e.g., such as hCds1, MTKs, PTK, vided in the kit. The diagnostic profile comprises meas- CDKs, STKs, CaMs, and the like) (see, e.g., United ured levels of theone or more peptides ina samplehaving States Patent No. 6,426,206); phosphatases; human the known cell state (e.g., a cell state corresponding to drug metabolizing proteins; viral proteins, including but a normal physiological response or to an abnormal phys- not limited to viral envelope proteins (e.g., an HIV enve- 50 iological response, such as a disease). lope protein); transporter proteins; and the like. [0123] Preferably, the software enables a processor [0117] In one aspect, the peptide internal standard to receive a plurality of diagnostic profiles and to select comprises a label associated with a modified amino acid a diagnostic profile that most closely resembles or residue, such as a phosphorylated amino acid residue, "matches" the profile obtained for the test cell state profile a glycosylated amino acid residue, an acetylated amino 55 by matching values of levels of proteins determined in acid residue, a famesylated residue, a ribosylated resi- the test sample to values in a diagnostic profile, to identify due, and the like. In another aspect, a pair of reagents substantially all of a diagnostic profile which matches the is provided, a peptide internal standard corresponding to test cell state profile.

15 27 EP 1 472 539 B1 28

[0124] Substantially all of a diagnostic profile is stantially the entire complement of proteins in a cell matched by a test cell state profile when most of the cel- or tissue. lular constituents (e.g., proteins in the proteome) which are diagnostic of the cell state, are found to have sub- 3. The method according to claim 1, wherein the pep- stantially the same value in the two profiles within a mar- 5 tide internal standard is labeled using a stable iso- gin provided by experimental error. Preferably, at least tope. about 75% of the diagnostic proteins can be matched, at least about 80%, at least about 85%, at least about 90% 4. The method according to claim 1, wherein the pres- or at least about 95% can be matched. Preferably, where ence and/or quantity of target polypeptide is diag- one, or only a few proteins (e.g., less than 10) are used 10 nostic of a cell state. to establish s diagnostic profile, preferably all of the proteins have substantially the same value. 5. The method according to claim 4, wherein the cell state is representative of an abnormal physiological response. Claims 15 6. The method according to claim 5, wherein the ab- 1. A method for determining the presence and/or quan- normal physiological response is diagnostic of a dis- tity of a target polypeptide in at least one mixture of ease. different polypeptides, comprising: 20 7. The method according to claim 4, wherein the cell a) providing a mixture of different polypeptides; state is a state of differentiation. b) adding a known quantity of a peptide internal standard labeled with a mass-altering label, 8. The method according to claim 1, wherein the first thereby generating a spiked mixture, wherein cell is a normal cell and the second cell is from a the labeled peptide internal standard comprises 25 patient with a disease. a subsequence of the target polypeptide and wherein the labeled peptide internal standard 9. The method according to claim 1, wherein the deter- possesses a known peptide fragment signature mining is done in parallel. diagnostic of the presence of the peptide; c) treating the spiked mixture with a protease 30 10. The method according to claim 1, wherein the two activity to generate a plurality of peptides includ- mixtures are the same and the labeled peptide inter- ing the labeled peptide internal standard and nal standard is provided in different known amounts peptides corresponding to the target polypep- in each mixture. tide; d) fragmenting by multistage mass spectrometry 35 11. The method according to claim 1, wherein the la- the labeled peptide internal standard and any beled peptide internal standard in each mixture com- target peptide present in the spiked mixture prises the same peptide but different labels. comprising the same amino acid sequence as the labeled peptide internal standard; e) determining the ratio of labeled fragments to 40 Patentansprüche unlabeled fragments; and f) calculating from the ratio and the known quan- 1. Verfahren zur Bestimmung des Vorliegens und/ oder tity of the labeled internal standard, the quantity der Menge eines Target-Polypeptids in wenigstens of the target polypeptide in the mixture; option- einem Gemisch verschiedener Polypeptide, umfas- ally 45 send: g) determining the presence and/or quantity of target peptides in at least two mixtures wherein (a) Bereitstellen eines Gemischs verschiedener one mixture is from a cell having a first cell state Polypeptide; and the other mixture is from a cell having a sec- (b) Zugeben einer bekannten Menge eines in- ond cell state. 50 ternen Peptid-Standards, der mit einer Massen- Änderungsmarkierung markiert ist, wodurch ein 2. The method according to claim 1, wherein the mix- gespiktes Gemisch hergestellt wird, wobei der ture of different polypeptides is selected from the markierte interne Peptid-Standard eine Unter- group consisting of: a crude fermenter solution, a folge des Target- Polypeptidsumfasst und wobei cell-free culture fluid, a cell or tissue extract, blood 55 der markierte interne Peptid-Standard eine be- sample, a plasma sample, a lymph sample, a cell or kannte Peptidfragment-Signatur besitzt, die für tissue lysate; a mixture comprising at least about 100 das Vorliegen des Peptids diagnostisch ist; different polypeptides; a mixture comprising sub- (c) Behandeln des gespikten Gemisches mit ei-

16 29 EP 1 472 539 B1 30

ner Proteaseaktivität, um eine Vielzahl von Pep- 9. Verfahren gemäß Anspruch 1, wobei die Bestim- tiden, die den markierten internen Peptid-Stan- mung parallel erfolgt. dard umfassen, und von Peptiden, die dem Tar- get-Polypeptid entsprechen, zu erzeugen; 10. Verfahren gemäß Anspruch 1, wobei die zwei Ge- (d) Fragmentieren durch Mehrstufen-Massen- 5 mische die selben sind und der markierte interne spektrometrie des markierten internen Peptid- Peptid-Standard in verschiedenen bekannten Men- Standards und eines Target-Peptids, das indem gen in jedem Gemisch bereitgestellt wird. gespikten Gemisch vorliegt, das die selbe Ami- nosäuresequenz wie der markierte interne Pep- 11. Verfahren gemäß Anspruch 1, wobei der markierte tid-Standard umfasst; 10 interne Peptid-Standard in jedem Gemisch das sel- (e) Bestimmen des Verhältnisses von markier- be Peptid, aber unterschiedliche Markierungen um- ten Fragmenten zu unmarkierten Fragmenten fasst. und (f) Berechnen aus dem Verhältnis und der bekannten Menge des markierten internen Stan- 15 Revendications dards der Menge des Target- Polypeptids in dem Gemisch; gegebenenfalls 1. Procédé de détermination de la présence et/ou de (g) Bestimmen des Vorliegens und/oder der la quantité d’un polypeptide cible dans au moins un Menge von Target- Peptiden in wenigstens zwei mélange de différents polypeptides, comprenant : Gemischen, wobei ein Gemisch aus einer Zelle, 20 die einen ersten Zellzustand hat, stammt und a) fournir un mélange de différents das andere Gemisch aus einer Zelle, die einen polypeptides ; zweiten Zellzustand hat, stammt. b) ajouter une quantité connue d’un étalon interne peptidique marqué avec un marqueur d’al- 2. Verfahren gemäß Anspruch 1, wobei das Gemisch 25 tération de masse, ce qui permet de générer un verschiedener Polypeptide ausgewählt wird aus der mélange enrichi, où l’étalon interne peptidique Gruppe, bestehend aus: einer rohen Fermenterlö- marqué comprend une sous- séquence du poly- sung, einer zellfreien Kulturflüssigkeit, einem Zell- peptide cible et où l’étalon interne peptidique oder Gewebeextrakt, einer Blutprobe, einer Plasma- marqué possède un diagnostic de signature du probe, einer Lymphprobe, einem Zell- oder Gewe- 30 fragment peptidique connu permettant de con- belysat; einem Gemisch, das wenigstens etwa 100 firmer la présence du peptide ; unterschiedliche Polypeptide umfasst; einem Ge- c) traiter le mélange enrichi avec une activité de misch, das im Wesentlichen das gesamte Komple- protéase pour produire une pluralité de peptides ment von Proteinen in einer Zelle oder einem Gewe- comprenant l’étalon interne peptidique marqué be umfasst. 35 et des peptides correspondant au polypeptide cible ; 3. Verfahren gemäß Anspruch 1, wobei der interne d) fragmenter par spectrométrie de masse multi- Peptid-Standard unter Verwendung eines stabilen étape l’étalon interne peptidique marqué et un Isotops markiert ist. peptide cible quelconque présent dans le mé- 40 lange enrichi comprenant la même séquence 4. Verfahren gemäß Anspruch 1, wobei das Vorliegen d’acides aminés que l’étalon interne peptidique und/oder die Menge an Target-Polypeptid für einen marqué ; Zellzustand diagnostisch ist. e) déterminer le ratio entre les fragments mar- qués et les fragments non marqués ; et 5. Verfahren gemäß Anspruch 4, wobei der Zellzu-45 f) calculer à partir du ratio et de la quantité con- stand für eine abnormale physiologische Reaktion nue d’étalon interne peptidique marqué, la repräsentativ ist. quantité de polypeptide cible dans le mélange ; éventuellement 6. Verfahren gemäß Anspruch 5, wobei die abnormale g) déterminer la présence et/ou la quantité de physiologische Reaktion für eine Krankheit diagno- 50 peptides cibles dans au moins deux mélanges stisch ist. où un mélange provient d’une cellule ayant un premier état cellulaire et l’autre mélange pro- 7. Verfahren gemäß Anspruch 4, wobei der Zellzu- vient d’une cellule ayant un deuxième état cel- stand ein Differenzierungszustand ist. lulaire. 55 8. Verfahren gemäß Anspruch 1, wobei die erste Zelle 2. Procédé selon la revendication 1, dans lequel le mé- eine normale Zelle ist und die zweite Zelle von einem lange de différents polypeptides est choisi dans le Patienten mit einer Krankheit ist. groupe constitué par : une solution de fermenteur

17 31 EP 1 472 539 B1 32

brute, un fluide de culture acellulaire, un extrait cellulaire ou tissulaire, un échantillon de sang, un échantillon de plasma, un échantillon de lymphe, un lysat cellulaire ou tissulaire ; un mélange comprenant au moins environ 100 polypeptides différents ; 5 un mélange comprenant sensiblement le complé- ment entier de protéines dans une cellule ou dans un tissu.

3. Procédé selon la revendication 1, dans lequel l’éta- 10 lon interne peptidique est marqué en utilisant un isotope stable.

4. Procédé selon la revendication 1, dans lequel la pré- sence et/ou la quantité de polypeptide cible est le 15 diagnostic d’un état cellulaire.

5. Procédé selon la revendication 4, dans lequel l’état cellulaire est représentatif d’une réponse physiologique anormale. 20

6. Procédé selon la revendication 5, dans lequel la ré- ponse physiologique anormale est le diagnostic d’une maladie. 25 7. Procédé selon la revendication 4, dans lequel l’état cellulaire est un état de différentiation.

8. Procédé selon la revendication 1, dans lequel la pre- mière cellule est une cellule normale et la deuxième 30 cellule provient d’un patient ayant une maladie.

9. Procédé selon la revendication 1, dans lequel la dé- termination est effectuée en parallèle. 35 10. Procédé selon la revendication 1, dans lequel les deux mélanges sont identiques et l’étalon interne peptidique marqué est prévu en différentes quantités dans chaque mélange. 40 11. Procédé selon la revendication 1, dans lequel l’éta- lon interne peptidique marqué dans chaque mélange comprend le même peptide mais différents mar- queurs. 45

18 EP 1 472 539 B1

19 EP 1 472 539 B1

20 EP 1 472 539 B1

21 EP 1 472 539 B1

22 EP 1 472 539 B1

23 EP 1 472 539 B1

24 EP 1 472 539 B1

25 EP 1 472 539 B1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 0125884 W [0048] • US 6303297 B [0103] • US 6426206 B [0064] [0116]

Non-patent literature cited in the description

• Computational Molecular Biology. Oxford University • Pearson ; Lipman. Proc. Natl. Acad. Sci. USA, Press, 1988 [0040] 1988, vol. 85, 2444 [0040] • Biocomputing: Informatics and Genome Projects. Ac- • Altschul et al. Nucleic Acids Res., 1997, vol. 25 (17), ademic Press, 1993 [0040] 3389-3402 [0040] • Computer Analysis of Sequence Data. Humana • E. Myers ; W. Miller. CABIOS, 1989, vol. 4, 11-17 Press, 1994 [0040] [0040] • von Heinje, G. Sequence Analysis in Molecular Bi- • James ; Wilkins. Anal. Chem., 1990, vol. 62, ology. Academic Press, 1987 [0040] 1295-1299 [0058] • Sequence Analysis Primer. M Stockton Press, 1991 • Williams et al. J Amer. Soc. Mass Spectrom., 1990, [0040] vol. 1, 413-416 [0058] • Needleman ; Wunsch. J. Mol. Biol., 1970, 444-453 • Zubarev et al. J. Am. Chem. Soc., 1998, vol. 120, [0040] 3265-3266 [0058] • Smith ; Waterman. Adv. Appl. Math., 1981, vol. 2, • Kern et al. Int. J. Oncol., 2001, vol. 21 (2), 243-9 482 [0040] [0093]