WO2007140352A2

WO2007140352A2 - Plasma membrane and secreted cancer biomarkers

Info

Publication number: WO2007140352A2
Application number: PCT/US2007/069825
Authority: WO
Inventors: Xiquan Liang; Robert Pope; Mahbod Hajivandi; John Leite; Christopher Adams; Lisa Wenrich
Original assignee: Invitrogen Corporation
Priority date: 2006-05-26
Filing date: 2007-05-26
Publication date: 2007-12-06
Also published as: WO2007140352A3

Abstract

Plasma membrane and secreted protein biomarkers are identified for breast cancer, in which the biomarkers are proteins having about a two-fold or greater difference in abundance between breast cancer and normal cells. Identified biomarkers can be used in detection methods that can provide diagnosis, monitoring, typing, staging, or prognosis of cancer, such as breast cancer, or can be used to predict the response of a cancer, such as a breast cancer, to one or more therapeutic regimens, for example, treatment with anti-cancer agents, radiation, etc.

Description

PLASMA MEMBRANE AND SECRETED CANCER BIOMARKERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to United States provisional application 60/808,809, filed May 26, 2006, entitled "Plasma Membrane and Secreted Cancer Biomarkers" and naming Xiquan Liang, Robert M. Pope, Mahbod Hajivandi, and John Leite as inventors; which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a "lengthy" Sequence Listing which has been submitted electronically, and is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

[0003] The invention relates generally to biomarkers for cancer, and more specifically to biomarkers for breast cancer, and to the detection of biomarker expression and abundance in cancer cells.

Background Information

[0004] Quantitative proteomics, techniques used to measure differential expression and processing profiles of the entire complement of gene products, may be used to identify and characterize onset disease biomarkers by comparing cell culture samples from both normal and disease states. Stable isotope labeling with amino acids in cell culture (SILAC) is an emerging technology for quantitative proteomics, allowing quantification of the cellular differences between two different states. SILAC methods and applications are described in U.S. Patent No. 6,391,649 and U.S. Patent No. 6,642,059, both of which are herein incorporated by reference in their entireties, and in particular for all disclosure of methods of labeling proteins of cells in culture with isotopes and comparing protein levels of the cell cultures using mass spectrometry (MS). [0005] SILAC uses the natural metabolic machinery of the cell to label proteins with either 'light' or 'heavy' amino acids made with light (standard) or heavy isotopes. Peptides with either light or heavy amino acids are chemically identical and therefore co-migrate in any separation method (such as SDS-PAGE, IEF or other liquid chromatography, etc.), eliminating quantification error due to unequal sampling. However, the peptides are isotopically distinct so that the mass difference between light and heavy peptides is distinguishable by mass spectrometry (MS). Based on the relative intensity of an isotopic peptide pair in MS, differential protein expression and the status of posttranslational modification between two different samples can be quantified. The correlation of a particular peptide to the precursor protein from which it originates is based upon the fragmentation pattern of the (usually mass-selected) peptide, hence its MS/MS profile.

[0006] SILAC offers additional advantages over other technologies such as ease of use, compatibility with any lysis buffer or separation technology, and 100% labeling efficiency. Because the incorporation of stable isotopic forms of amino acids occurs as the proteins are assembled or degraded in cell culture, these chemically identical proteins are copies of their light congeners bearing what are, in effect, "label-less labels" at every site of the substituted amino acid. Moreover, since one is free to select any amino acid as a label, one may select an amino acid specific to any protease used in a digestion protocol later and, thus achieve a single label on each and every digest fragment. This makes it possible to track the status of posttranslational modifications, because in principle, proteome coverage is complete.

[0007] MicroRNAs are a recently discovered class of small, -19-23 -nucleotide non-coding RNA molecules. They are cleaved from 70-110-nucleotide hairpin precursors and are believed play an important role in translation regulation and degradation of target mRNAs by binding to partially complementary sites in the 3' untranslated regions (UTRs) of the message (Lim, L. P., Glasner, M. E., Yekta, S., Burge, C. B., Bartel,D. P. (2003). Science 299, 1540.). Recent experimental evidence suggests that the number of unique miRNAs in humans could exceed 800, though several groups have hypothesized that there may be up to 20,000 non-coding RNAs that contribute to eukaryotic complexity (Bentwich et al, (2005) Nature Genetics, 37: 766-770).

[0008] Though hundreds of miRNAs have been discovered in a variety of organisms, little is known about their cellular function. They have been implicated in processes such as regulation of developmental timing and pattern formation (Lagos-Quintana et al. (2001) Science 294: 797- 799) restriction of differentiation potential (Nakahara & Carthew (2004) Curr. Opin. Cell Biol. 16: 127-33.), and genomic rearrangements (John et al. (2004) Am. J. Hum. Genet. 75: 54-64).

[0009] Several unique physical attributes of miRNAs — including their small size, lack of poly-adenylated tails, and tendency to bind their mRNA targets with imperfect sequence homology — have made them elusive and challenging to study. In addition, strong conservation between miRNA family members means that any detection technology must be able to distinguish between ~22-base sequences that differ by only 1-2 nucleotides. Recent advances in spotted oligonucleotide microarray labeling and detection have enabled the use of this high- throughout technology for miRNA screening.

SUMMARY OF THE INVENTION

[00010] The present invention expands the use of SILAC to the detection of biomarkers that can be useful in the detection and classification of disease state cells, such as cancer cells. The present invention provides methods of identifying biomarkers using SILAC, methods of detecting and classifying cells using biomarkers identified by SILAC, and biomarkers useful in the detection and classification of cancer, particularly breast cancer.

[00011] The present invention also provides methods for detection of miRNAs in cells, such as cancer cells, where increased levels of miRNAs can correlate with decreased levels of proteins whose expression is decreased in cancer cells relative to normal cells.

[00012] The present invention provides methods of identifying a multiplicity of proteins whose levels differ among cells of a disease state and normal cells using stable isotope labeling of cells in culture, and using one or more of the identified proteins as biomarkers for a disease state. The present invention provides reliable methods of identifying markers that are differentially expressed in one or more cell compartments, such as, for example, cell membranes. The present invention also provides methods of identifying markers that are differentially secreted from one cell type when compared with another cell type, such as, for example, cancer cells versus normal cells.

[00013] In one aspect, the present invention includes a method of identifying at least one biomarker for cells of a disease state, where the method includes: providing a first cell culture of disease state cells; providing a second cell culture of control cells, in which the cell culture media of the first cell culture contains at least one isotope at a non-natural level in a form that is metabolically incorporated into proteins within cultured cells, in which the isotope is present at a natural level is the second cell culture; allowing the cells in each of the cell cultures to divide; combining at least a portion of the media or cells of the first cell culture with at least a portion of the media or cells of the second cell culture to form a mixed cell or mixed cell media sample; separating one or more proteins from the mixed cell or mixed cell media sample; performing mass spectrometry on the one or more proteins, or peptides generated from the proteins, to obtain a mass spectrometry profile; using the mass spectrometry profile to compare the abundance of at least one of the one or more proteins containing the isotope at a natural level with the abundance of the one or more corresponding proteins that contain the isotope at a non-natural level, in which a difference in abundance of a protein having the isotope at a non-natural level and the same protein having the isotope at a natural level is indicative of a biomarker for disease state cells.

[00014] In some preferred embodiments, disease state and normal cells are fractionated prior to the separation of proteins for mass spectrometry. For example, cells can be fractionated for the isolation of cell membranes. Proteins from cell fractions can be further separated, such as by electrophoresis or chromatography, preferably digested with a protease, and analyzed using mass spectrometry to identify proteins that have different abundances in disease state and normal cells. United States Patent Application serial number 11/417,264 entitled "Identification of Cancer Biomarkers and Phosphorylated Proteins" naming Pope, Liang, Hajivandi, and Leite as inventors and filed on May 4, 2006 is herein incorporated by reference in its entirety for all purposes, including for all disclosure of methods of culturing cells in light and heavy isotopes for comparison of protein abundance in different cell cultures, cell fractionation and protein enrichment and separation methods, and for disclosure of identification of peptides and protein using mass spectrometry, and comparison of protein abundances in cultures using mass spectrometry.

[00015] Cell media can also be used for the identification of proteins that may be secreted by cancer cells in greater amounts than by normal cells. Using methods disclosed herein, secreted proteins can be concentrated from the media of cancerous and normal cells grown in culture, further fractionated, and identified and analyzed for their relative abundances in the media of cancerous and normal cells.

[00016] The invention provides methods for detecting one or more proteins as provided herein (for example, the proteins listed in Table 1 and Table 2) that are differentially expressed in cancer cells and normal cells of the same type. The invention also provides methods for detecting one or more nucleic acids encoding a protein as provided herein (for example, a protein listed in Table 1 or Table 2) that is differentially expressed in cancer cells and normal cells of the same type. In one aspect, the invention provides methods of detecting the abundance of a protein of Table 1 or Table 2 in or on a cancer cell, a precancerous cell, a premalignant cell, or a cell exhibiting an atypia, or in cancer tissue or a precancerous or pre-malignant lesion, or in bodily fluids of a patient or cultured from a patient having or at risk of or suspected of having cancer, a pre-malignancy, a precancerous lesion, or a cell atypia. In some embodiments, the invention provides methods of detecting the abundance of a protein of Table 1 or Table 2 in or on a cancer cell, premalignant cell, precancerous cell, or cell exhibiting an atypia; or in or on cancerous, precancerous, or premalignant tissue or tissue exhibiting an atypia; or in bodily fluids of a patient having cancer or a malignancy, or having a precancerous or premalignant lesion or a cell atypia, in which the abundance of the protein in or on a cell, tissue, or bodily fluid of a patient differs from that of a normal cell or tissue of the same type, or differs from that of bodily fluid of a patient not exhibiting cancer, a malignancy, a premalignancy or precancerous lesion, or a cell atypia.

[00017] The present invention also provides biomarkers for cancer. Such biomarkers can be used to detect or diagnose cancer in a subject, such as a patient suspected of having cancer, as well as to screen patients who may not be suspected to have cancer but are nonetheless at risk. One or more than one of the identified biomarkers can be used to detect, diagnose, type, stage, provide a prognosis for, or predict a treatment (e.g., drug, radiation, etc.) response of cancer in a patient. One or more than one of the identified biomarkers can be used to detect, diagnose, type, stage, provide a prognosis for, or predict a drug response of breast cancer in a patient.

[00018] Data provided herein identifies the proteins of Table 1 as being plasma membrane proteins or proteins associated with the cell surface that are differentially expressed, or differentially secreted or released from the cell surface by human breast cancer cells. Accordingly, provided herein is a method of detecting one or more biomolecules, comprising detecting in a biological sample, the presence or abundance of a protein of Table 1, or a nucleic acid encoding a protein of Table 1, wherein the biological sample is a sample of a patient with a cancer. The cancer can be of any type, such as but not limited to, lung cancer, pancreatic cancer, colon cancer, uterine cancer, ovarian cancer, prostate cancer, a leukemia, a lymphoma, or breast cancer. The biological sample can be, for example, a tumor biopsy sample or a breast tumor biopsy sample. Furthermore, the sample can be a fluid sample. For example, sample of blood, plasma, serum, urine, saliva, cerebrospinal fluid, lymphatic fluid, pelvic lavage, lung aspirate, mucus, sputum, nipple aspirate, or breast duct lavage.

[00019] Data provided herein identifies the proteins of Table 2 as being secreted proteins or proteins released from the cell surface that are differentially expressed, or differentially secreted or released from the cell surface by human breast cancer cells. Accordingly, provided herein is a method of detecting one or more biomolecules, comprising detecting in a biological sample, the presence or abundance of a protein of Table 2, or a nucleic acid encoding a protein of Table 2, wherein the biological sample is a sample of a patient with a cancer. The cancer can be of any type, such as but not limited to, lung cancer, pancreatic cancer, colon cancer, uterine cancer, ovarian cancer, prostate cancer, a leukemia, a lymphoma, or breast cancer. The biological sample can be, for example, a tumor biopsy sample or a breast tumor biopsy sample. Furthermore, the sample can be a fluid sample. For example, sample of blood, plasma, serum, urine, saliva, cerebrospinal fluid, lymphatic fluid, pelvic lavage, lung aspirate, mucus, sputum, nipple aspirate, or breast duct lavage.

[00020] In certain aspects of the invention, expression is detected of two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty- five or more, one-half, one -third, or all of the proteins listed in Table 1. In certain aspects of the invention, expression is detected of two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty- five or more, one-half, one -third, or all of the proteins listed in Table 2. In certain aspects of the invention, expression is detected of two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty- five or more, one-half, one-third, or all of the proteins listed in Table 1 and Table 2, or of nucleic acids encoding at least a portion of two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty-five or more, one-half, one-third, or all of the proteins of Table 1, Table 2, or Table 1 and Table 2.

[00021] In certain aspects of the invention, expression is detected of one or more miRNAs that regulate the expression of one or more, two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty- five or more, one- half, one-third, or all of the proteins listed in Table 1. In certain aspects of the invention, expression is detected of one or more "micro RNA molecules" or miRNAs that regulate the expression of one or more, two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty-five or more, one-half, one- third, or all of the proteins listed in Table 2. In certain aspects of the invention, expression is detected of one or more miRNAs that regulate the expression of two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty- five or more, one-half, one-third, or all of the proteins listed in Table 1 and Table 2. [00022] Certain aspects of the invention provide quantitative methods. For example, in certain aspects an expression level is determined of a protein of Table 1 or Table 2, or a nucleic acid encoding at least a portion of a protein of Table 1 or Table 2, or an miRNA that regulates the expression of at least one protein of Table 1 or Table 2. In certain embodiments, an altered expression level of a protein, mRNA, or miRNA in the biological sample compared to a normal sample is indicative of the presence of cancer or a breast pathology, such as breast cancer. Furthermore, expression levels can be correlated with a type of cancer, a stage of cancer or a precancerous state, a prognosis, and/or response to one or more anti-cancer agents.

[00023] Typically, methods of this embodiment of the invention are performed by contacting the biological sample with a specific binding member that binds to the protein or the nucleic acid molecule. Expression levels can then be quantitated by measuring the amount of specific binding member that binds to biomolecules in the sample.

[00024] In another embodiment, provided herein is a kit that includes a specific binding member that binds to a protein of Table 1 or Table 2, or that binds to a nucleic acid encoding a protein of Table 1 or Table 2. In some embodiments, the kits include two or more specific binding members that bind to a protein of Table 1 or Table 2, or that bind to a nucleic acid encoding a protein of Table 1 or Table 2. Furthermore, the kits can optionally include a positive control designed to confirm that an assay performed using the materials provided in the kit functioned as intended. Reagents for performing any suitable positive control can be included. For example, the positive control could be one or more partially or substantially purified proteins that react with one or more specific binding members of the kit, or can be a biological sample derived from a subject having a breast pathology. For example, the control can include cells obtained directly from a subject having a breast pathology, or tissue culture cells derived from cells of a subject having a breast pathology, such as breast cancer or a breast tumor. The specific binding member of the kit is typically an antibody, antibody derivative (e.g., a fragment of an antibody, which fragment retains at least one antigen-binding portion of an antibody), or a nucleic acid. The specific binding member is typically present in one or more tubes that are associated together in packaging and shipped from a manufacturer to a customer. The kit can include additional specific binding members. For example, specific binding members that bind to one or more, two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty-five or more, one half, one third, or all of the proteins of Table 1 or Table 2, or nucleic acid molecules encoding at least a portion of a protein of Table 1 or Table 2.

[00025] In one embodiment, the kit includes specific binding members that bind to one or more, two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty- five or more, one -half, one-third, or all of the proteins of Table 1 or Table 2, or nucleic acid molecules encoding at least a portion of a protein of Table 1 or Table 2.

[00026] The invention also provides kits that include one or more nucleic acid molecules that hybridize to one or more miRNAs that regulate the expression of any of the proteins of Table 1 or Table 2.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic depiction of some embodiments of the present invention for relative quantification of membrane proteins between normal and malignant breast cell lines originating from the same patient with breast carcinoma.

Figure 2 depicts a tryptic membrane protein peptide with light and heavy labels analyzed by mass spectrometry. Multiple isotopic peptide pairs derived from platelet derived growth factor receptor were recovered in a single LC-MS/MS run demonstrating the precision of quantification.

Figure 3 depicts MS spectra demonstrate the reproducibility of SILAC experiments. SILAC experiments were performed three times separately. Spectra for a CTLC protein peptide detected in the three experiments are shown.

Figure 4 depicts MS spectra of several proteins obtained using the membrane protein isolation procedures provided herein found to be (A) downregulated in breast cancer cells and (B) upregulated in breast cancer cells.

Figure 5 is a schematic depiction of methods of identifying secreted proteins having different levels of expression in normal and malignant breast cell lines originating from the same patient with breast carcinoma.

Figure 6 depicts mass spectra of four peptides of fibronectin precursor in the same experiment that analyzed proteins released into the media of breast cancer and normal cells showing the precision of quantitation.

Figure 7 depicts mass spectra showing the results of quantitation of the same peptide in three separate experiments that analyzed protein secreted into the media by breast cancer cells and normal cells.

Figure 8 shows (A) MALDI-TOF mass spectra of peptides of osteoblast specific factor 2, a protein having greater abundance in the media of breast cancer cells as compared with the media of normal cells and Serpin E2, a protein having reduced abundance in the media of breast cancer cells as compared with the media of normal cells and (B) the corroborating Western blot data. DETAILED DESCRIPTION OF THE INVENTION

[00027] The present invention is based on the discovery that biomarkers can be identified by using SILAC to analyze changes in membrane and secreted protein expression in disease state cells, including primary cultured cells. Furthermore, the present invention is based in part on the identification of a set of new cancer, particularly breast cancer, biomarkers, using the methods provided herein.

I. Identification of Plasma Membrane Biomarkers

[00028] The present invention uses methods for identifying at least one plasma membrane biomarker for cells of a disease state by comparing the levels of one or more membrane proteins in two or more cell cultures using stable isotope labeled amino acids in cell culture. These methods comprise: (1) providing a first cell culture comprising disease state cells; (2) providing a second cell culture comprising normal cells, in which the cell culture media of the first cell culture comprises at least one isotope at a non-natural abundance, and the second cell culture does not comprise the at least one isotope at a non-natural abundance, where the isotope at a non- natural abundance is in a from that is metabolically incorporated into proteins within cultured cells; and (3) allowing the cells in each of the cell cultures to divide. After the cells have divided in culture so that isotopes in the culture media have been incorporated into proteins, the method further comprises: (4) labeling the surfaces of the cells with a protein-labeling reagent; (5) combining at least a portion of the cells of the first cell culture with at least a portion of the cells of the second cell culture to form a mixed cell sample; (6) lysing the cells under conditions that preserve membrane structure; (7) separating one or more membrane fragments proteins from the lysed cell sample using an affinity reagent that binds the protein labeling reagent; (8) isolating proteins from the membrane fragments; (9) performing mass spectrometry on the one or more proteins, or peptides derived from one or more proteins, to obtain a mass spectrometry profile; and (10) using the mass spectrometry profile to compare the level of at least one of the one or more proteins comprising the isotope at a non-natural abundance with the level of the at least one of the one or more proteins that does not comprise the isotope at a non-natural abundance, in which a difference in the level of the non-naturally occurring isotope form of a protein from the naturally occurring isotope form is indicative of a biomarker for cells of a disease state. [00029] In these methods, membrane fragments or "rafts" are isolated by affinity capture of cell surface proteins labeled with a moiety such as, for example, biotin. The labeling of cells can occur before or after aliquots of two or more cell cultures are mixed. For example, an aliquot of normal cells cultured in light isotope media can be surface-labeled, with, for example a biotin label that attached to proteins, and an aliquot of cancer cells of the same type grown in heavy media can be cultured in heavy isotope media and separately cell surface-labeled prior to mixing the two cell types, lysing the cells, and performing affinity capture and protein isolation. In an alternative procedure, cells of the two cultures can be mixed prior to cell surface labeling.

[00030] Preferably, the second culture comprises an isotope at a much lower abundance than is present in the first culture, for example, the first culture can comprise an isotope at a level that is much greater than its natural abundance, and the second culture can comprise the isotope at a level that is equivalent to the isotopes natural abundance, or can 10% or less of its level in the first cell culture, 5% or less of its level in the first cell culture, 2% or less of its level in the first cell culture, or 1% or less of its level in the first cell culture. In some embodiments of the invention, the first cell culture can comprise an isotope at a level that is greater than its natural abundance, and the second cell culture can comprise the isotope at a level that is not detectable such as by mass spectrometry to detect incorporation of isotopes into protein.

[00031] The present application incorporates by reference U.S. Patent No. 6,391,649 and U.S. Patent No. 6,642,059 in their entireties, including SILAC methods and applications and for methods of labeling proteins of cells in culture with isotopes and comparing protein levels of the cell cultures using mass spectrometry (MS). This application also incorporates by reference United States Patent Application serial number 11/417,264, entitled, "Identification of Cancer Biomarkers and Phosphorylated Proteins" naming Pope, Liang, Hajivandi, and Leite as inventors and filed on May 4, 2006, in its entirety, including disclosure of methods for comparing protein levels of two or more cell cultures and identification of biomarkers using isotope labeling of cell cultures and mass spectrometry. In preferred embodiments, the methods are performed using two cell cultures that preferably comprise cells of the same type, where one culture comprises normal cells and the other culture comprises cells of a disease state. In some preferred applications of the method, the cells can be normal and cancer cells of the same type (for example, normal breast cells and breast cancer cells). [00032] Any affinity reagent that can label proteins can be used for cell surface labeling. Moieties that can be affinity captured can be derivatized using methods known in the art, so that they can covalently bind proteins, such as through lysine, cysteine, or other reactive amino acids. Labeling of cell surface proteins is preferably followed by lysis of cells under conditions that do not disrupt membranes. By "preserve membrane structure" is meant that although membranes may be fragmented, they are not solubilized, and that integral membrane proteins and proteins covalently bound to membrane lipids remain with the membranes. Using the cell lysis procedures herein, such as lysis in hypotonic buffers and/or physical disruption, membrane fragments or "rafts" are captured using affinity reagents that bind the cell surface label. One example of a hypotonic lysis buffer is 10 mM Tris, pH 7.4, 1 mM MgCl₂, 10 units per milliliter benzonase, 0.5 mM PMSF, 0.15 micromolar aprotinin, and 1 micromolar leupeptin hemisulfate.

[00033] Other membrane proteins, such as but not limited to integral membrane proteins, are captured as well by virtue of their association with the membrane. Washes of affinity captured reagents can be of variable stringency to remove proteins from the periphery of membranes. Wash stringencies can be done, for example, at various salt concentrations and various pH's to optimize conditions according to the researcher's goals. In some exemplary methods, salt conditions can range from about 50 mM to about 2 M. The pH of a wash solution can vary widely, from below to well above neutrality. In some exemplary methods, the pH of a wash solution can vary from about 7 to about 14, or from about 7.5 to about 13.

[00034] An alternative method for identifying at least one cell surface plasma membrane biomarker for cells of a disease state comprises: (1) providing a first cell culture comprising disease state cells; (2) providing a second cell culture comprising normal cells, in which the cell culture media of the first cell culture comprises at least one isotope at a non-natural abundance, and the second cell culture does not comprise the at least one isotope at a non-natural abundance, where the isotope at a non-natural abundance is in a from that is metabolically incorporated into proteins within cultured cells; and (3) allowing the cells in each of the cell cultures to divide. After the cells have divided in culture so that isotopes in the culture media have been incorporated into proteins, the method further comprises: (4) labeling the surfaces of the cells with a protein- labeling reagent; (5) combining at least a portion of the cells of the first cell culture with at least a portion of the cells of the second cell culture to form a mixed cell sample; (6) lysing the cells under conditions that solubilize membranes; (7) separating one or more membrane proteins from the lysed cell sample using an affinity reagent that binds the protein labeling reagent; (8) performing mass spectrometry on the one or more proteins, or peptides derived from one or more proteins, to obtain a mass spectrometry profile; and (10) using the mass spectrometry profile to compare the level of at least one of the one or more proteins comprising the isotope at a non-natural abundance with the level of the at least one of the one or more proteins that does not comprise the isotope at a non-natural abundance, in which a difference in the level of the non-naturally occurring isotope form of a protein from the naturally occurring isotope form is indicative of a biomarker for cells of a disease state.

[00035] In these methods, proteins that are exposed to the cell surface are specifically labeled and captured. The labeling of cells can occur before or after aliquots of two or more cell cultures are mixed. For example, an aliquot of normal cells cultured in light isotope media can be surface- labeled, with, for example a biotin label that attached to proteins, and an aliquot of cancer cells of the same type grown in heavy media can be cultured in heavy isotope media and separately cell surface-labeled prior to mixing the two cell types, lysing the cells using methods that solubilize cell membranes, and performing affinity capture and protein isolation. One example of a buffer for cell lysis contains 50 mM Tris-HCl, pH 8, 1% NP-40, 150 mM NaCl, 1 mM Na₃VO₄, 10 mM NaF, 0.15 micromolar aprotinin, and 1 micromolar leupeptin hemisulfate. Another example of a buffer for cell lysis contains 50 mM Tris-HCl, pH 8, 1% TritonX-100, 0.5% deoxycholate, 0.1% SDS, 500 mM NaCl, 1 mM Na₃VO₄, 10 mM NaF, 0.15 micromolar aprotinin, and 1 micromolar leupeptin hemisulfate.

[00036] In an alternative procedure, cells of the two cultures can be mixed prior to cell surface labeling.

[00037] Figure 1 outlines protocols for applying the methods of the present invention to identifying plasma membrane biomarkers in cancer cells. In this depiction, normal and cancer cells are grown in media containing either "light" lysine (Ly s) and arginine (Arg), that have naturally occurring isotopes of, for example nitrogen and carbon, or "heavy" Lys and Arg, that have a higher abundance of an isotope of nitrogen or carbon, for example, than the natural abundance. In both methods, the two cultures are incubated for at least six doubling times and then cell surface-labeled with biotin. The cells are then combined at 1 : 1 ratio.

[00038] In the method depicted in the panel on the left in Figure 1, the cell mixture is lysed in hypotonic buffer and membrane rafts are captured using streptavidin beads. The membrane rafts captured to the beads are then washed (for example, with high salt washes followed by high pH washes) before being solubilized in detergent to remove integral membrane proteins. The membrane proteins are precipitated and dissolved in SDS sample buffer and analyzed by SDS- PAGE. The entire gel lane is divided into approximately 30-50 sections, followed by in-gel tryptic digestions. Peptide extracts are analyzed by mass spectrometry (such as, but not limited to, nanoelectrospray LC-MS/MS). Relative quantification is achieved via the ratios of unique isotopic peptide pairs in the resulting MS spectrum.

[00039] In the method depicted in the panel on the right in Figure 1 , the cell mixture is lysed in a detergent buffer and cell surface proteins labeled with biotin are captured using streptavidin beads. The beads are washed and then the cell surface membrane proteins are removed from the beads using a denaturing detergent (for example, SDS) and analyzed by SDS-PAGE. The entire gel lane is divided into approximately 40 sections, followed by in-gel tryptic digestions. Peptide extracts are analyzed by mass spectrometry (such as, but not limited to, nanoelectrospray LC- MS/MS). Relative quantification is achieved via the ratios of unique isotopic peptide pairs in the resulting MS spectrum.

[00040] The two methods can be complementary. For example, while the two isolation methods may identify many of the same proteins, it is also the case that some integral membrane proteins (as well as proteins covalently bound to membrane lipids) can be identified by capturing membrane rafts that may not be identified by cell surface labeling, if the membrane proteins are not exposed on the cell surface, or do not have efficiently labeled amino acids exposed on the cell surface. On the other hand, some cell surface labeled proteins may be captured after denaturing the cell membranes, but may be lost by high salt or high pH washes of captured membrane rafts. The methods disclosed herein can be used in combination to identify a large number of plasma membrane-associated proteins that would not identified using a single isolation method. II. Identification of Secreted Biomarkers

[00041] The present invention uses methods for identifying at least one secreted biomarker for cells of a disease state by comparing the levels of one or more membrane proteins in two or more cell cultures using stable isotope labeled amino acids in cell culture. These methods comprise: (1) providing a first cell culture comprising disease state cells; (2) providing a second cell culture comprising normal cells, in which the cell culture media of the first cell culture comprises at least one isotope at a non-natural abundance, and the second cell culture does not comprise the at least one isotope at a non-natural abundance, where the isotope at a non-natural abundance is in a from that is metabolically incorporated into proteins within cultured cells; and (3) allowing the cells in each of the cell cultures to divide. After the cells have divided in culture so that isotopes in the culture media have been incorporated into proteins, the method further comprises: (4) repaleing the media of each of the cell cultures with fresh media; (5) incubating the cells in fresh media for a period of time; (6) combining at least a portion of the media of the first cell culture with at least a portion of the media of the second cell culture to form a mixed cell culture media sample; (7) isolating one or more proteins from the combined media sample; (9) performing mass spectrometry on the one or more proteins, or peptides derived from one or more proteins, to obtain a mass spectrometry profile; and (10) using the mass spectrometry profile to compare the level of at least one of the one or more proteins comprising the isotope at a non-natural abundance with the level of the at least one of the one or more proteins that does not comprise the isotope at a non-natural abundance, in which a difference in the level of the non-naturally occurring isotope form of a protein from the naturally occurring isotope form is indicative of a biomarker for cells of a disease state.

[00042] In these methods, secreted proteins also include proteins that are released into the media by means other than the cellular secretory pathway. For example, a protein or a fragment of a protein can be released from the cell surface of cancer cells even though it is typically not a "secreted" proteins.

[00043] In preferred embodiments, after a sufficient labeling period in which one cell culture divides in light isotope cell media, and another cell culture divides in heavy isotope cell media, the media of the cell cultures is exchanged for fresh media (not containing heavy isotope) and after a specific length of time, for example, from fifteen minutes to twenty-four hours later, the cell media is harvested for analysis.

[00044] Preferably, a concentration step is performed to concentrated proteins prior to their isolation from the collected cell media. Other isolation or separation steps can optionally be performed prior to analysis of the proteins, such as, but not limited to, affinity purification.

[00045] Preferably, the second culture comprises an isotope at a much lower abundance than is present in the first culture, for example, the first culture can comprise an isotope at a level that is much greater than its natural abundance, and the second culture can comprise the isotope at a level that is equivalent to the isotopes natural abundance, or can 10% or less of its level in the first cell culture, 5% or less of its level in the first cell culture, 2% or less of its level in the first cell culture, or 1% or less of its level in the first cell culture. In some embodiments of the invention, the first cell culture can comprise an isotope at a level that is greater than its natural abundance, and the second cell culture can comprise the isotope at a level that is not detectable such as by mass spectrometry to detect incorporation of isotopes into protein.

[00046] The present application incorporates by reference U.S. Patent No. 6,391,649 and U.S. Patent No. 6,642,059 in their entireties, including SILAC methods and applications and for methods of labeling proteins of cells in culture with isotopes and comparing protein levels of the cell cultures using mass spectrometry (MS). This application also incorporates by reference United States Patent Application serial number 11/417,264, entitled, "Identification of Cancer Biomarkers and Phosphorylated Proteins" naming Pope, Liang, Hajivandi, and Leite as inventors and filed on May 4, 2006, in its entirety, including disclosure of methods for comparing protein levels of two or more cell cultures and identification of biomarkers using isotope labeling of cell cultures and mass spectrometry. In preferred embodiments, the methods are performed using two cell cultures that preferably comprise cells of the same type, where one culture comprises normal cells and the other culture comprises cells of a disease state. In some preferred applications of the method, the cells can be normal and cancer cells of the same type (for example, normal breast cells and breast cancer cells).

[00047] Figure 5 provides a schematic diagram of methods for identifying proteins secreted or released by cancer cells and normal cells, and to determine their abundance in cell media. Cells

[00048] The cells used in the methods of the present invention can be prokaryotic or eukaryotic cells of any type, and are preferably animal cells, and more preferably are mammalian cells. Cells used in the methods of the present invention are most preferably human cells.

[00049] The cells used for identifying biomarkers can be from cell lines or primary cells. The inventors have found that using the methods of the present invention, it is possible to use as few as 10⁶ cells to identify differential expression between two cell types or two cell states. For optimal labeling of cells, the cells are preferably grown in heavy isotope media for at least six doublings to allow for greater than 98% incorporation of label. Thus, starting cultures can have as few as 2 x 10⁵ cells. This allows for the use of primary cells, such as cells from lines that have not been immortalized through genetic manipulation or have not otherwise become growth factor-independent. This also allows for the use of primary cell isolated directly from a subject, such as cells from tissue samples (including biopsy samples) in which the cell number is limited, to determine differences in protein expression in normal and disease state cells using SILAC methods. Primary cells can be taken from biopsied or sampled tissue or bodily fluids, and are preferably at least partially purified away from other sample components, including other cell types ("nontarget cells"), using, for example, the use of separation steps such as filtration, centrifugation, or selective precipitation; dissection of tissue (including but not limited to laser capture microdissection); affinity separation of components (such as by "panning" using affinity reagents such as antibodies directly or indirectly bound to a solid support such as beads to either remove undesirable sample components or enrich cells of interest); or the application of drugs or reagents to a culture that discourage the growth of nontarget cells.

[00050] In cases where primary cells are used, proteins expressed by primary disease-state cells can be compared in SILAC experiments with proteins expressed normal cells of a cell line, but preferably are compared with primary normal cells. The primary normal cells can be from the same or a different individual. Proteins expressed by primary normal cells can also be compared with proteins expressed by disease state cells of a cell line. Isotopic Label

[00051] The isotopic labels used in the methods of the present invention can be any molecule that can be metabolically incorporated into protein within cells that has a non-natural abundance of one or more isotopes. As nonlimiting examples, heavy isotopes of carbon, nitrogen, oxygen, hydrogen, and sulfur that are of very low abundance in nature can be highly enriched in molecules used to label proteins (such as amino acids) such that, using mass spectrometry, proteins or peptides that have incorporated the heavy isotopes can be distinguished from corresponding proteins or peptides that have incorporate isotopes of the same element at their natural abundance, that is, "light" isotopes of the element.

[00052] Preferred labels are amino acids having non-naturally occurring levels of heavy isotopes, such as, but not limited to, carbon-13, nitrogen-15, oxygen-17, oxygen-18, sulfur-34, and hydrogen-2. An amino acid can be labeled with more than one isotope at a non-natural abundance, for example, an amino acid used in SILAC can have both carbon-13 and nitrogen-15. For a given cell labeling/MS detection experiment, one or more amino acids can be labeled with a non-natural abundance of one or more isotopes. In some preferred embodiments of the present invention, for example, Arg and Lys have incorporated heavy isotopes (such as ¹³C and/or ¹⁵N), and proteins are digested with trypsin prior to mass spectrometry. In these embodiments, each trypsin fragment of a protein of the labeled cell culture (with the exception of carboxy-terminal peptides) comprises a heavy isotope label.

[00053] In the methods of the present invention, protein levels of two cultures are compared by comparing heavy isotope-labeled proteins or peptides of one culture with light isotope containing proteins or peptides of the other culture. Preferably, in these methods, one of the cultures comprises a heavy isotope label, where the heavy isotope is at a non-natural abundance, and the other culture does not comprise an isotope label at a non-natural abundance. For example, the first culture can comprise in the media a metabolic precursor molecule that can become incorporated into biomolecules such as proteins within cells, in which the metabolic precursor molecule comprises an isotope at a non-natural abundance, and the second culture can comprise in the media the same metabolic precursor molecule, in which the precursor molecule does not comprise an isotope at a non-natural abundance. The metabolic precursor molecule can be, as nonlimiting examples, a sugar or amino acid. The isotope present at a non-natural abundance can be, for example a heavy isotope.

[00054] The heavy isotope label can be present in either the control cell culture or in the culture of disease state cells. In preferred embodiments in which the label is an amino acid that comprises one or more heavy isotopes, preferably the heavy isotope amino acid comprises essentially all of the amino acid in the cell culture to be labeled with heavy isotope. For example, where the label is ¹³C -Arg, preferably the culture media of the culture to be labeled contains ¹³C -Arg to the exclusion of ¹²C -Arg. Preferably, the cell culture that does not comprise a heavy isotope label at a non-natural abundance does not contain a detectable level of the heavy isotope.

Culturing Cells

[00055] Normal and disease state cells are preferably cultured in parallel, in which either the normal cell culture media or the disease state cell culture media comprises a label in the form of an isotope at non-natural abundance. In some preferred embodiments, the normal cell culture media comprises a label in the form of an isotope at non-natural abundance, and the disease state culture media does not comprise a label in the form of an isotope at non-natural abundance. In other preferred embodiments, the disease state cell culture media comprises a label in the form of an isotope at non-natural abundance, and the normal cell culture media does not comprise a label in the form of an isotope at non-natural abundance. In other preferred embodiments, both cell cultures whose cells are being compared comprise isotopic label, where one cell culture comprises a first isotopic label, and the second cell culture comprises a second isotopic label.

[00056] In other embodiments, cells can be removed from a tissue, such as but not limited to a cancerous tissue such as a tumor, and grown in culture with an isotopic label. The cells can be combined with cells of the original tumor from patient biopsy for MS analysis to compare the abundance of proteins expressed by tumor cells grown in culture with the abundance of the same proteins expressed by tumor cells in the body. The comparison can be used to identify biomarkers for tumor cells that relate to the ability of the tumor to survive and grow within the body of a patient, such as but not limited to biomarkers that participate in the interaction of cancer cells with normal cells, such as biomarkers related to, for example, tissue infiltration, tumor vascularization, and nutrient procurement. Such biomarkers can be candidate drug targets. [00057] Thus, the present invention provides a method of identifying proteins that enable, mediate, or facilitate tumor growth in the body. The method includes: providing a culture comprising cancer cells removed a tumor from a patient; allowing the tumor cells in the cell culture to divide in media that comprises at least one isotope at a non-natural level in a form that is metabolically incorporated into proteins within cultured cells; combining at least a portion of the cells of the cell culture with a sample of cells taken directly from the tumor to form a mixed cell sample; separating one or more proteins from the mixed cell sample; performing mass spectrometry on the one or more proteins, or peptides thereof, to obtain a mass spectrometry profile; and using the mass spectrometry profile to compare the abundance of at least one of the one or more proteins comprising the non-naturally occurring isotope with the abundance of the at least one of the one or more proteins that does not comprise the non-naturally-occurring isotope. In these methods, a difference in the abundance of the non-naturally occurring isotope form relative to the naturally occurring isotope form is indicative of a protein that enables, facilitates, or mediates the growth of tumor cells in the body.

[00058] Preferably the label is a heavy isotope of, for example, carbon, nitrogen, sulfur, oxygen, or hydrogen that is incorporated into an amino acid in the cell culture media used for labeling cells. Preferably the cell culture media used for labeling cells comprises one or more heavy isotope-labeled amino acids, in which greater than 95%, and even more preferably greater than 98%, of the one or more amino acids that are labeled comprise the heavy isotope.

[00059] For example, cells that are to be labeled with one or more particular amino acids that have one or more incorporated heavy isotopes can be grown in DMEM, RPMI, or any other suitable media to which the one or more heavy isotope amino acids have been added. Parallel cultures in which the proteins are not to be labeled with heavy isotope amino acids preferably are supplemented with the same amino acids as the labeled cultures, but in this case the amino acids do not comprise heavy isotopes. Preferably, the media and media supplements used to culture the cells do not contain the particular amino acids that are to be supplied to the cultures in heavy isotope form for labeling. For example, serum used for culturing the cells should be dialyzed to remove amino acids. [00060] As nonlimiting examples, cells to be labeled can be grown in DMEM or RPMI medium to which dialyzed FBS has been added to a final concentration of 10%. The media can be supplemented with glutamine (where glutamine is not used as a labeled amino acid), and, optionally, pencillin and/or streptomycin at standard concentrations. Purified growth factors or cytokines can be supplemented to the media if they are essential to cell growth or desirable for the experiment being performed.

[00061] In these examples, the labeling media also contains 100 mg of at least one heavy isotope amino acid, such as, for example, [U-¹³C₆] L-Lysine, [U-¹³C₆] L-Arginine, or [U-¹⁵N₄, U-¹³C₆,] L-Arginine. (In one preferred embodiment, the media contains 100 mg/liter heavy Lys ([U-¹³C₆] L-Lysine) and 100 mg/liter heavy Arg ([U-¹⁵N₄, U-¹³C₆,] L-Arginine), such that peptides containing heavy Lys experience a shift of 6 Da relative to their unlabeled counterparts, and peptides containing heavy Arg experience a shift of 10 Da relative to their unlabeled counterparts.) The corresponding non-labeling media is supplemented with the same amounts of the same amino acids as the labeling media, but in this case in their light isotope form. For example, where the labeling media is supplemented with 100 mg per liter of each of heavy Arg and heavy Lys, the non-labeling media is supplemented with 100 mg per liter of each of non- heavy isotope Arg and non-heavy isotope Lys.

[00062] Cell are preferably grown in labeling media for at least six doublings. For example, a starting culture of 10⁵ cells can be grown to a final cell number of 6.4 x 10⁶ cells. This ensures essentially 100% incorporation of heavy isotope amino acids into proteins of the cells. Depending on the growth rate of the cells and the culture density, the cells can be split with light or heavy labeling medium separately.

[00063] To ensure 100% incorporation of heavy amino acids into proteins, small aliquots of cells (10⁵-10⁶) labeled with light or heavy amino acids can be removed and lysed separately in 500 μl of SDS sample buffer and analyzed by SDS-PAGE side by side. One or two protein bands are picked randomly, excised from the gel side by side, and subjected to in-gel tryptic digest, followed by the MALDI-TOF analysis. Alternatively, cells (10⁷) labeled with light or heavy amino acids can be lysed in cell lysis buffer separately. Proteins of interest can then be immunoprecipitated from the cell lysates and analyzed by SDS-PAGE side by side. Protein bands are excised, digested with trypsin, and then analyzed by MALDI-TOF in parallel. Compared to peptides labeled with light amino acids, peptides labeled with heavy amino acids should increase a few Daltons in mass depending on the nature of the heavy amino acids used for labeling (e.g. 6 Da for ¹³C labeled Lys and 10 Da for ¹³C, ¹⁵N double-labeled Arg). If only heavy ¹³C Lys is used for labeling, only peptides containing a Lys residue will shift 6 Da in mass, but peptides containing an Arg residue would have the same mass. In this way, before proceeding with an experiment, close to 100% incorporation of heavy amino acid into peptides can be verified, which means that no or a very little of corresponding light peptides should be detected in a peptide sample from cells labeled with heavy amino acids.

Mixing of samples

[00064] In most applications of the method control cells and a disease state cells are mixed prior to performing any cell fractionation or protein separation steps. The mixing of control and disease state cells prior to further manipulation avoids sample -to-sample variation in the downstream steps leading to mass spectrometry that can lead to error in calculating relative abundances of proteins in the cultures being compared. In these procedures, aliquots of the two cell cultures being compared are preferably counted and equal numbers of control and disease- state cells are mixed together to form a mixed cell sample.

[00065] However, it may be desirable in some circumstances to mix cell organelles, cell lysates, extracts, or fractions after control and normal cell cultures have been separately lysed, and, optionally, subjected to one or more fractionation or separation steps. In these cases, equal amounts of the lysates, extracts, or fractions can be mixed after quantitating or measuring activity of one or more cellular components.

[00066] In methods of the present invention in which cell media is analyzed to detect secreted proteins, the cell supernatants are mixed. Preferably, aliquots of each cell culture are counted so that the amount of cell supernatants of the two cell cultures that are mixed together is standardized to the number of cells in each culture. It is also possible in this case to mix cell supernatants of each cell culture based on equal protein content of the cell supernatants, or equal amounts or activities of one or more components the cell supernatants. [00067] Many membrane proteins mediate the response of the cell to external factors, such as growth factors, hormones, other cells, and cell substrates. Therefore, the separation of cell membranes for investigating differences in abundance of membrane proteins between normal cells and disease state cells is of particular interest. In addition to serving as biomarkers, membrane proteins identified as being differentially expressed in a disease state can be candidate drug targets.

[00068] Proteins isolated from cell media or cell fractions can be separated to reduce the complexity of the samples analyzed by mass spectrometry. Separation of proteins can be, for example, by affinity capture, selective solubilization, selective precipitation, chromatography, or electrophoresis. Affintiy capture can be used to separate a single protein or protein family, epitope tagged proteins, or a broad class or proteins, such as, for example, proteins containing phosphotyrosine. Chromatography can be affinity chromatography, or can separate proteins based on size, charge, or hydrophobicity. Chromatographic separation can be coupled to mass spectrometry, as described below, to sequentially analyze fractions as they elute from a column matrix. Electrophoresis, such as PAGE, can be used to separate proteins based on size. PAGE can be under denaturing or nondenaturing conditions, or two-dimensional PAGE can be performed. After electrophoresis, a gel lane comprising separated proteins can be divided into slices. Proteins extracted from each of the slices or any subset of the slices can be analyzed separately using mass spectrometry.

[00069] For identification of proteins using SILAC, the proteins are preferably digested into peptides prior to mass spectrometry. Proteins can be digested using any protease or chemical peptide cleavage reagent that generates peptides of from about 5 to about 200 amino acids. Examples of proteases that can be used include trypsin, V-8 protease, pepsin, subtilisin, proteinase Ic, and tobacco etch virus protease. Cyanogen bromide can also be used. Trypsin is preferred in some embodiments of the present invention in which arginine and lysine are isotopically labeled amino acids. In these embodiments, because trypsin digests proteins C- terminal to arg and lys residues, each trypsin fragment (except for carboxy terminal fragments of proteins) will have an isotopic label. [00070] A general tryptic digest protocol is as follows:

[00071] To avoid contamination with keratin, gel manipulation and digestion steps are performed in a laminar flow hood. Protein bands of interest are excised, along with positive and negative controls, and at least one blank from the SDS-PAGE gel. NuPAGE® Novex® acrylamide gels, available from Invitrogen (Carlsbad, CA, USA) are recommended for protein separation. Each band/or spot is chopped into small (approx lmm diameter) particles with a clean pipette tip. The gel pieces are transferred to a clean microcentrifuge tube. Microtubes from Axygen (Union City, CA) or Eppendorf (Hamburg, Germany) are recommended The gel pieces are destained two to three times with 40% acetonitrile in 25 mM NH4HCO3 (pH 8.0) until no blue hue is observed. The gel pieces are dehydrated with 100% acetonitrile and dried for 5 min with a Speed Vac lyophilizer (Savant).

[00072] About 10 μl of cold 10 ng/ml proteomics-grade trypsin in 25 mM NH₄HCO₃ (pH 8.0) is added to the dried gel pieces. The gel pieces are incubated on ice for 1 to 2 hr as they swell to minimize auto proteolysis as the trypsin soaks into the gel. Just enough trypsin solution is added to cover the gel pieces. The microcentrifuge tube is covered with aluminum foil to ensure uniform heating, and the gel pieces are incubated overnight at 37⁰C.

[00073] Approximately 30 μl of 1.5% TFA is added to the tryptic digestion mix and incubated at room temperature for about 30 min. The tubes are vortexed about 1 min, centrifuged briefly, and the peptide extract is transferred into a clean tube (Axygen or Eppendorf microcentrifuge tubes are recommended). The gel pieces are further extracted with 30 to 50 μl of 50% acetonitrile in 0.75% TFA for 30 min. and vortexed for about 1 min. The peptide extracts are combined and analyzed by MALDI-TOF. For LC-ESI/MS analysis, the peptide extract should be dried under vacuum and the peptides resuspended in 20 μl of 10% acetonitrile in 0.1% formic acid. Alternatively, 2% formic acid can be used in place of TFA in the steps above. Mass Spectrometry

[00074] In various aspects, the invention is drawn to mass spectroscopy. As used herein, the term "mass spectrometry" (or simply "MS") encompasses any spectrometric technique or process in which molecules are ionized and separated and/or analyzed based on their respective molecular weights. Thus, as used herein, the terms "mass spectrometry" and "MS" encompass any type of ionization method, including without limitation electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI) and other forms of atmospheric pressure ionization (API), and laser irradiation. Mass spectrometers are commonly combined with separation methods such as gas chromatography (GC) and liquid chromatography (LC). GC or LC separates the components in a mixture, and the components are then individually introduced into the mass spectrometer; such techniques are generally called GC/MS and LC/MS, respectively. MS/MS is an analogous technique where the first-stage separation device is another mass spectrometer. In LC/MS/MS, the separation methods comprise liquid chromatography and MS. Any combination (e.g., GC/MS/MS, GC/LC/MS, GC/LC/MS/MS, etc.) of methods can be used to practice the invention. In such combinations, "MS" can refer to any form of mass spectrometry; by way of non- limiting example, "LC/MS" encompasses LC/ESI MS and LC/MALDI-TOF MS. Thus, as used herein, the terms "mass spectrometry" and "MS" include without limitation APCI MS; ESI MS; GC MS; MALDI-TOF MS; LC/MS combinations; LC/MS/MS combinations; MS/MS combinations; etc.

HPLC and RP-HPLC

[00075] It is often necessary to prepare samples comprising an analyte of interest for MS. Such preparations include without limitation purification and/or buffer exchange. Any appropriate method, or combination of methods, can be used to prepare samples for MS. One preferred type of MS preparative method is liquid chromatography (LC), including without limitation HPLC and RP-HPLC.

[00076] High-pressure liquid chromatography (HPLC) is a separative and quantitative analytical tool that is generally robust, reliable and flexible. Reverse-phase (RP) is a commonly used stationary phase that is characterized by alkyl chains of specific length immobilized to a silica bead support. RP-HPLC is suitable for the separation and analysis of various types of compounds including without limitation biomolecules, (e.g., glycoconjugates, proteins, peptides, and nucleic acids, and, with mobile phase supplements, oligonucleotides). One of the most important reasons that RP-HPLC has been the technique of choice amongst all HPLC techniques is its compatibility with electrospray ionization (ESI). During ESI, liquid samples can be introduced into a mass spectrometer by a process that creates multiple charged ions (WiIm et al., Anal. Chem. 68:1, 1996). However, multiple ions can result in complex spectra and reduced sensitivity.

[00077] In HPLC, peptides and proteins are injected into a column, typically silica based C 18. An aqueous buffer is used to elute the salts, while the peptides and proteins are eluted with a mixture of aqueous solvent (water) and organic solvent (acetonitrile, methanol, propanol). The aqueous phase is generally HPLC grade water with 0.1% acid and the organic solvent phase is generally an HPLC grade acetonitrile or methanol with 0.1% acid. The acid is used to improve the chromatographic peak shape and to provide a source of protons in reverse phase LC/MS. The acids most commonly used are formic acid, triflouroacetic acid, and acetic acid. In RP HPLC, compounds are separated based on their hydrophobic character. With an LC system coupled to the mass spectrometer through an ESI source and the ability to perform data- dependant scanning, it is now possible in at least some instances to distinguish proteins in complex mixtures containing more than 50 components without first purifying each protein to homogeneity. Where the complexity of the mixture is extreme, it is possible to couple ion exchange chromatography and RP-HPLC in tandem to identify proteins from mixtures containing in excess of 1,000 proteins.

MALDI-TOF MS

[00078] A particular type of MS technique, matrix-assisted laser desorption time-of- flight mass spectrometry (MALDI-TOF MS) (Karas et al., Int. J. Mass Spectrom. Ion Processes 78:53, 1987), has received prominence in analysis of biological polymers for its desirable characteristics, such as relative ease of sample preparation, predominance of singly charged ions in mass spectra, sensitivity and high speed. MALDI-TOF MS is a technique in which a UV-light absorbing matrix and a molecule of interest (analyte) are mixed and co-precipitated, thus forming analyte matrix crystals. The crystals are irradiated by a nanosecond laser pulse. Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. Nevertheless, matrix molecules transfer their energy to analyte molecules, causing them to vaporize and ionize. The ionized molecules are accelerated in an electric field and enter the flight tube. During their flight in this tube, different molecules are separated according to their mass to charge (m/z) ratio and reach the detector at different times. Each molecule yields a distinct signal. The method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides and oligonucleotides, with molecular masses between about 400 and about 500,000 Da, or higher. MALDI-MS is a sensitive technique that allows the detection of low (10^~15 to 10^"18 mole) quantities of analyte in a sample.

[00079] Partial amino acid sequences of proteins can be determined by enzymatic proteolysis followed by MS analysis of the product peptides. These amino acid sequences can be used for in silico examination of DNA and/or protein sequence databases. Matched amino acid sequences can indicate proteins, domains and/or motifs having a known function and/or tertiary structure. For example, amino acid sequences from an uncharacterized protein might match the sequence or structure of a domain or motif that binds a ligand. As another example, the amino acid sequences can be used in vitro as antigens to generate antibodies to the protein and other related proteins from other biological source material (e.g., from a different tissue or organ, or from another species). There are many additional uses for MS, particularly MALDI-TOF MS, in the fields of genomics, proteomics and drug discovery. For a general review of the use of MALDI- TOF MS in proteomics and genomics, see Bonk et al. (Neuroscientist 7:12, 2001).

[00080] Tryptic peptides labeled with light or heavy amino acids can be directly analyzed using MALDI-TOF. However, where sample complexity is apparent, on-line or off-line LC- MS/MS or two-dimensional LC-MS/MS is necessary to separate the peptides. For example, for simple digests, a gradient of 5-45% (v/v) acetonitrile in 0.1% formic acid (or TFA, if MALDI MS/MS is available) over 45 min, and then 45-95% acetonitrile in 0.1% formic acid (or TFA, if MALDI MS/MS is available) over 5 min can be used. 0.1% Formic acid solution is used on the Q-TOF instrument and 0.1% TFA solution is used on the Dionex Probot fraction collector for off-line coupling between HPLC and MALDI-MS/MS analysis (carried out on the ABI 4700). For a complex sample, a gradient of 5-45% (v/v) acetonitrile over 90 min, and then 45-95% acetonitrile over 30 min is used. For a very complex sample, a gradient of 5-45% (v/v) acetonitrile over 120 min, and then 45-95% acetonitrile over 60 min might be used. On the Q- TOF, one survey scan and four MS/MS data channels are used to acquire CID data with 1.4 s scan time. On the 4700 proteomics, the most intense eight peptides with mass over 1000 are chosen for MS/MS analysis.

Identification of Biomarkers

[00081] Software programs such as MSQuant can be used for quantification of protein expression (msquant.sourceforge.net). See, for example, Olsen et al. (2006) Cell 127: 635-648 for an example of the use of a quantification program.

[00082] Biomarkers are identified as proteins having an abundance in disease state cells that is either greater than or less than that in normal cells, or having an abundance in a given cellular compartment or fraction of disease state cells that is greater or less than that in the same cellular compartment or fraction of normal cells. The amount by which the abundance of a protein can differ between disease state and normal cells to be identified as a biomarker, for example, can be greater than 20%, greater than 30%, greater than 50%, greater than 70%, greater than 90%, or greater than 100%. For example, a biomarker can be an identified protein whose abundance in disease state cells is at least about 200% (or about 2-fold) greater than or less than its abundance in normal cells. In other cases, a biomarker can be an identified protein whose abundance in disease state cells is at least about 300% (or about 3 -fold) greater than or less than its abundance in normal cells. In yet other cases, a biomarker can be an identified protein whose abundance in disease state cells is at least about 500% (or about 5 -fold) greater than or less than its abundance in normal cells.

[00083] A protein identified as a biomarker can be a previously characterized protein or a protein that has not been previously characterized. Preferably, mass spectrometry analysis provides amino acid sequence of peptides of proteins that differ in abundance between normal and disease state cultures, and such amino acid sequences can be compared with nucleic acid and protein sequence databases. Where antibodies are unavailable for characterized or uncharacterized proteins, they can be generated using methods known in the art using synthetic peptides or recombinant or purified protein. Such antibodies can be used to validate biomarkers as well as for detection of biomarkers in tissues samples.

[00084] An advantage of using SILAC/MS to identify biomarkers of disease state cells is that the method provides an extensive, and, in principle, complete profile of the proteins expressed by cells that belong to the class of proteins targeted in the separation methods (for example, integral membrane proteins, secreted proteins). Thus, using the methods of the present invention users can identify multiple biomarkers for a disease state. The identification of multiple biomarkers can allow for more reliable detection methods, where cells of a disease state can be identified, and potentially classified, by analysis of expression levels of multiple proteins.

[00085] Biomarkers can be validated by confirming expression differences between normal and disease state cells using methods of detecting protein levels other than SILAC. For example, protein level comparisons can be performed using immunocytochemistry, Western blot, immunoprecipitation, ELISA, or other antibody binding and detection methods. Biomarkers can also be validated using methods of detecting nucleic acids that encode at least a portion of a biomarker protein. Such methods can include FISH, CISH, polymerase based assays, nucleic acid array hybridization and hybridization assays such as but not limited to Northern blot analysis as they are known in the art.

III. Use of Biomarkers in Detecting Protein Expression in Disease State Cells [00086] Biomarkers for a disease state identified by the methods disclosed herein can be used to detect expression of proteins in cells of a disease state or pre-disease state, such as, but not limited to, cancer cells or precancerous cells. For example, as disclosed above, disease state cells such as cancer cells and normal cells of the same type can be grown in parallel cultures, in which either the disease state cell culture or the normal cell culture contains one or more heavy isotope amino acids. After growth of cells in culture, such that essentially all of the protein in the heavy isotope culture is labeled, equal numbers of the cells can be mixed, and the cells can be subjected to cell fractionation, protein separation, and protein digestion. Peptides resulting from protein digestion of the pooled cell culture samples can be analyzed by mass spectrometry and, preferably, multiple biomarkers can be identified in which the biomarker is present at different level in disease state cells and normal cells. [00087] In further experiments, one or more of the identified biomarkers can be detected in one or more biological test samples, such as tissue samples or bodily fluid samples. The sample can be a tumor biopsy sample, a blood, plasma, or serum sample, lymphatic fluid, saliva, a lung aspirate, a nipple aspirate, breast duct lavage sample, a pelvic lavage sample, a swab or scraping, etc. The sample need not be of the same tissue that was used to identify the biomarkers. For example, biomarkers that are overexpressed in cancer cells relative to normal cells that are localized on the cell membranes of cancer cells may be detected in the blood or lymph, and thus blood, plasma, serum, and lymphatic fluid can be used to detect a disease state by detecting the presence of one or more biomarkers. Fluid samples used to harvest cells, cell fragments, and proteins at or near the site of a tumor (such as aspirates or lavages) can also be samples for detecting one or more biomarkers. The data obtained by determining the relative or absolute abundance of biomarkers in a sample can aid in the diagnosis of a disease state. In some preferred embodiments, the biological test sample is a biological sample from a subject know to have or suspected of having breast cancer, a breast neoplasm, a precancerous breast lesion, or a breast premalignancy, and the sample can be, for example, a serum sample, a breast biopsy sample, a nipple aspirate, or a breast duct lavage sample.

[00088] The present invention also includes methods of detecting one or more biomarkers expressed by a cancer cell, where the biomarker is a protein of Table 1 or Table 2, or a nucleic acid encoding at least a portion of a protein of Table 1 or Table 2, comprising detecting in a biological sample of a patient known to have or suspected of having cancer, a neoplasm, a precancerous lesion, or premalignant cells, an expression level of a protein of Table 1 or Table 2, or a nucleic acid encoding the protein of Table 1 or Table 2, wherein an altered expression level in the biological sample compared to the expression level in a normal sample is indicative of a cancerous or pre-cancerous state. A patient can be known to have or suspected of having, for example, breast cancer, uterine cancer, ovarian cancer, pancreatic cancer, colon cancer, lung cancer, prostate cancer, a leukemia, or a lymphoma. A patient or subject known to have or suspected of having a neoplasm, cancer, a precancerous lesion, or premalignant cells, can have one or more indicators of cancer or premalignancy as determined by family history (including genetic tests), personal history (including environmental exposure, diet, and physical symptoms), or diagnostic procedures, such as but not limited to, an X-ray, mammogram, ultrasound, histological morphology of biopsied tissue, or detection based tests, such as but not limited to, immunohistochemistry of biopsied tissue, or detection of different biomarkers (e.g., CA 125) from serum or other tissue samples (such as biopsied tissue).

[00089] Biomarkers identified by the methods of the present invention are not limited to proteins having the database sequences of the identified biomarker protein, but also include proteins encoded by the same gene at the same chromosomal locus as the identified biomarker. Thus, detecting a biomarker of Table 1 or Table 2 also includes detecting a protein encoded by allelic variants of those identified as encoding the proteins listed in Table 1 or Table 2, or protein variants resulting from one or more mutations or from alternative splicing of the encoding gene, or proteins differing from the proteins listed in Table 1 or Table 2 in post-translational modifications, including but not limited to proteolytic processing.

[00090] Data provided herein identifies the proteins of Table 1 and Table 2 as being proteins that are differentially expressed in or differentially secreted or released by human breast cancer cells. Accordingly, provided herein is a method of detecting one or more biomolecules, comprising detecting in a biological sample, expression of a protein of Table 1 or Table 2, or a nucleic acid encoding at least a portion of a protein of Table 1 or Table 2, wherein said biological sample is a sample of a patient with a breast pathology, such as a neoplasm, tumor, or precancerous lesion. The biological sample can be, for example, a tumor biopsy sample or a breast tumor biopsy sample. Furthermore, the sample can be a fluid sample. For example, sample of blood, plasma, serum, urine, saliva, lymphatic fluid, pelvic lavage, lung aspirate, nipple aspirate, or breast duct lavage.

[00091] Also provided herein is a method of detecting an miRNA, such as but not limited to miR Iet7d (SEQ ID NO: 112), miR 17-5p (SEQ ID NO: 113); miR 20-a (SEQ ID NO: 114); miR 21 (SEQ ID NO: 115); miR-30b (SEQ ID NO: 116); miR-106a (SEQ ID NO: 117); miR-106b (SEQ ID NO:118); or miR-195 (SEQ ID NO:119). An elevated level of one or more miRNAs can be indicative of a breast pathology, such as breast cancer. Methods of detecting miRNAs are known in the art, and can employ, for example, nucleic acid hybridization techniques, such as array technology.

[00092] In certain aspects of the invention, expression is detected of two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty- five or more, one-half, one -third, or all of the proteins of Table 1 and/or Table 2, or of nucleic acids encoding two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty-five or more, one-half, one- third, or all of the proteins of Table 1 and/or Table 2.

[00093] Certain aspects of the invention provide quantitative methods. For example, in certain aspects an expression level is determined of a protein of Table 1 or Table 2, or a nucleic acid encoding a protein of Table 1 or Table 2. In certain embodiments, an altered expression level in the biological sample compared to a normal sample (or control value based on expression in a sample of normal cells or tissue) is indicative of the presence of a breast pathology, such as breast cancer. For example, elevated expression, relative to a normal value obtained in the same detection methods from noncancerous biological samples, of one or more biomarkers of Table 1, or one or more biomarkers of Table 2, or one or more biomarkers of Table 1 in combination with one or more biomarkers of Table 2, can be correlated with the presence of cancer, a neoplasm, or a precancerous state in a subject. In another example, reduced expression of one or more biomarkers of Table 1, or one or more biomarkers of Table 2, or one or more biomarkers of Table 1 in combination with one or more biomarkers of Table 2, can be correlated with the presence of cancer, a neoplasm, or a precancerous state in a subject. In a further example, reduced expression of one or more biomarkers of Table 1, or one or more biomarkers of Table 2, or one or more biomarkers of Table 1 in combination with one or more biomarkers of Table 2, combined with increased expression of one or more biomarkers of Table 1 , or one or more biomarkers of Table 2, or one or more biomarkers of Table 1 in combination with one or more biomarkers of Table 2, can be correlated with the presence of cancer, a neoplasm, or a precancerous state in a subject. Furthermore, expression levels can be correlated with a type of cancer, a stage of cancer, a prognosis, and/or response to one or more anti-cancer agents.

[00094] Typically, methods of this embodiment of the invention are performed by contacting the biological sample with a specific binding member that binds to the protein or the nucleic acid. Expression levels can then be quantitated by measuring the amount of specific binding member that binds to biomolecules in the sample. The specific binding reagent is typically an antibody or a nucleic acid. In certain examples, the antibody can bind a secondary modification of a protein of Table 1 or Table 2. It will be recognized that the detection method can be an immunoassay.

[00095] In another embodiment, provided herein is a kit that includes a specific binding member that binds to a protein of Table 1 or Table 2, or that binds to a nucleic acid encoding a protein of Table 1 or Table 2. Furthermore, the kits typically include a positive control, such as may be derived from a biological sample taken from a subject having a breast pathology. For example, the control can include cells obtained directly from a subject having a breast pathology, or tissue culture cells derived from cells of a subject having a breast pathology, such as breast cancer or a breast tumor. The specific binding reagent of the kit is typically an antibody or a nucleic acid. The specific binding reagent is typically present in one or more tubes that are associated together in packaging and shipped from a manufacturer to a customer.

[00096] The kit can include additional specific binding reagents. For example, a kit can include specific binding members that bind to one or more, two or more, three or more, four or more, five or more, six or more, seven of more, eight or more, nine or more, ten or more, twenty- five or more, one-half, one -third, or all of the proteins, or encoding nucleic acids of Table 1 or Table 2.

[00097] In some preferred embodiments, one or more of the proteins listed in Table 1 and/or Table 2, or one or more nucleic acids encoding one or more of the proteins of Table 1 and/or Table 2, is detected in a biological sample of a patient known to have or suspected of having breast cancer, in which an altered expression level in the biological sample of the patient compared to the expression level in a normal sample is indicative of breast cancer. In some preferred embodiments, one or more of the proteins listed in Table 1 and/or Table 2, or one or more nucleic acids encoding one or more of the proteins of Table 1 and/or Table 2, is detected in a biological sample of a patient known to have or suspected of having breast cancer, in which an altered expression pattern biological sample of the patient compared to the expression pattern in a normal sample is indicative of breast cancer. The altered expression pattern can be, as nonlimiting examples, a different abundance, subcellular localization, aggregation status, activity, or post-translational modification. Detection of one or more biomarkers to detect breast cancer can be detection of two or more biomarkers detection of three or more biomarkers, or detection of four or more biomarkers of Table 1 and/or Table 2. [00098] Detecting a biomarker of Table 1 or Table 2 also includes detecting a protein encoded at the same genetic locus as those identified for the biomarkers of Table 1 or Table 2, including proteins encoded by allelic variants of the genes identified as encoding the proteins listed in Table 1 or Table 2, or protein variants resulting from mutations or from alternative splicing, or proteins differing from the proteins listed in Table 1 or Table 2 in post-translational modifications, including but not limited to proteolytic processing.

[00099] A biological test sample, or a fraction or extract thereof, can be tested for the presence, absence, or amount of one or more biomarkers, where the presence, absence, or amount of one or more biomarkers detected is indicative of the presence of cancer in the patient. The detection of a biomarker can use a biomarker binding member that specifically binds a biomarker, such as, for example, an antibody, or can use a biomarker binding reagent such as a nucleic acid molecule or nucleic acid analog that can specifically bind at least a portion of a nucleic acid that encodes a biomarker. As will be appreciated, while antibodies, antibody derivatives, and nucleic acids represent preferred classes of biomarker binding reagents, any binding reagent that is specific for a particular biomarker can be employed in practicing the invention. Detection of the biomarker is by detection of a label that can be directly or indirectly bound to or can directly or indirectly bind a biomarker binding member. A biomarker binding member can comprise or be directly or indirectly bound to detectable labels as they are known in the art, including but not limited to, radioactive, fluorescent, luminescent, or colorimetric labels. It is also possible to directly or indirectly bind a signal generating molecule or system to a specific binding member that binds a biomarker. For example, enzymes such as, but not limited to luciferase can be directly or indirectly bound to a specific binding member. A detection step can optionally include the addition of further members, such as substrates or cofactors, that are required for signal generation. Biomarker detection reagents can also be designed such that they can be specifically bound by a labeled reagent during the detection procedure, as in "sandwich" hybridization. Nonlimiting examples of detection methods useful in the present invention include immunocytochemistry, Western blotting, ELISA, immunoprecipitation, protein array detection, and other methods that use specific binding reagents such as but not limited to antibodies, and methods that employ nucleic acid hybridization (such as, but not limited to, Northern blots, array hybridization, FISH) and polymerase based methods such as, but not limited to, RT-PCR. [000100] Disease state cells or biomarkers derived from disease state cells can be identified using immunocytochemistry using an antibody that specifically binds the biomarker. In other preferred embodiments, the biomarker can be used to detect cells by immunoprecipitation, ELISA, or Western blot of cells, sample fluid, cell supernatants, or lysates prepared from the tissue sample.

[000101] In some preferred embodiments, a biomarker can be detected by detecting the nucleic acid that encodes the biomarker. Nucleic acid hybridization can be used, for example, Northern blot, array hybridization, or FISH can detect and, preferably, quantify nucleic acids encoding one or more biomarkers of a disease state.

[000102] One or more concentration steps, separation steps, or purification steps can optionally be performed on a biological sample prior to biomarker detection using the sample. For example, cells can be pelleted from fluid samples, and the cells can be further analyzed, or, alternatively, the supernatant of a centrifuged fluid sample can be analyzed for the presence or amount of one or more biomarkers. Where immunocytochemistry or FISH is used to detect disease state biomarkers, the cells can be fixed and prepared for antibody or nucleic acid binding using methods known in the art.

[000103] Cells obtained from tissue samples or fluid samples can optionally be lysed, and optionally, further fractionated or processed for protein or nucleic acid detection, for example, using immunoprecipitation, ELISA, or Western blot, or using nucleic acid hybridization or incorporation of nucleotides into nucleic acid molecules that encode at least a portion of a biomarker.

[000104] Preferably but optionally, detection of one or more biomarkers is quantitative or at least somewhat quantitative. By "somewhat quantitative" is meant that absolute amounts of the biomarker may not be determined, but amounts of biomarkers are determined relative to a standard, such as, for example, a standard of signal intensity based on comparison with controls that can be, for example, samples of normal cells, tissues, or biological samples, or fractions thereof. For example, the intensity of sample cell staining using a biomarker detection reagent can be scaled to the intensity of staining of one or more control cells. In another example, the amount of detection reagent bound to components isolated from biological test samples can also be compared with the amount of detection reagent bound to control components to calibrate levels of one or more biomarkers in the test sample.

[000105] The detection of the presence, absence, amount, or expression pattern of one or more biomarkers in a tissue sample or sample of bodily fluid can be indicative of a disease state. The sample can be a tumor biopsy sample, a blood, plasma, or serum sample, cerebrospinal fluid, lymphatic fluid, saliva, a lung aspirate, a nipple aspirate, breast duct lavage sample, a pelvic lavage sample, a swab or scraping, etc. taken from a patient suspected of having or known to have cancer. The sample, or a fraction or extract thereof, can be tested for the presence, absence, or amount of one or more biomarkers, where the presence, absence, or amount of one or more biomarkers detected is indicative of the presence of cancer in the patient.

[000106] The detection of the presence, absence, amount, or expression pattern of one or more biomarkers in a tissue sample or sample of bodily fluid can be indicative of a type or stage of a disease. The sample can be a tumor biopsy sample, a blood, plasma, or serum sample, cerebrospinal fluid, lymphatic fluid, saliva, a lung aspirate, a nipple aspirate, breast duct lavage sample, a pelvic lavage sample, a swab or scraping, etc. taken from a patient suspected of having or known to have cancer. The sample, or a fraction or extract thereof, can be tested for the presence, absence, or amount of one or more biomarkers, where the presence, absence, or amount of one or more biomarkers has been correlated with a type or stage of cancer. In this case, the presence or amount of one or more biomarkers detected in a patient sample can determine the type or stage of cancer in the patient. For example, the detection of the expression level of one or more proteins of Table 1 or Table 2 in a biological sample of a patient with cancer can be indicative of a type or stage of cancer, such as, but not limited to breast cancer. The detection of the expression level of one or more proteins of Table 1 or Table 2 together with additional information, including a patient's history, symptoms, and additional medical analysis can be indicative of a type or stage of cancer, such as, but not limited to breast cancer.

[000107] The detection of the presence, absence, amount, or expression pattern of one or more biomarkers in a tissue sample or sample of bodily fluid can be indicative of a prognosis of a disease, the progression of a disease, or the response of a disease to particular therapies. The sample can be a tumor biopsy sample, a blood, plasma, or serum sample, cerebrospinal fluid, lymphatic fluid, saliva, a lung aspirate, a nipple aspirate, breast duct lavage sample, a pelvic lavage sample, a swab or scraping, etc. taken from a patient suspected of having or known to have cancer, as well as from subjects who may simply be at risk for developing cancer. For example, the methods of the invention can be used to assay biological samples from presumably healthy women , i.e., women free from any symptom or history of breast cancer, for instance, for the presence, or level, of one or more biomarkers correlated with cancer, for example, a breast cancer. The sample, or a fraction or extract thereof, can be tested for the presence, absence, amount, or expression pattern of one or more biomarkers, where the presence, absence, or amount of one or more biomarkers has been correlated with a prognosis or a response to anticancer agents of the cancer. In this case, the presence or amount of one or more biomarkers detected in a patient sample can determine a prognosis or predict a drug response of the patient. For example, the detection of the expression pattern of one or more proteins of Table 1 or Table 2 in a biological sample of a patient known to have or suspected of having cancer can be indicative of a prognosis or of cancer, such as, but not limited to breast cancer. The detection of the expression pattern of one or more proteins of Table 1 or Table 2 taken together with other medical information and test results can be indicative of a prognosis of cancer, such as, but not limited to breast cancer.

[000108] In other embodiments, detection of the expression pattern of one or more proteins of Table 1 and Table 2 in a biological sample of a patient with cancer, such as, but not limited to breast cancer, can be used to predict a response to anti-cancer agents, such as chemotherapeutic drugs, anti-cancer monoclonal antibodies, hormones, etc, as well as therapeutic regimens such as surgery and/or radiation therapy, alone or in conjunction with one or more anti-cancer agents. In further embodiments, the detection of the expression level of one or more proteins of Table 1 and Table 2 can be used, in combination with information obtained from other tests or from the patient's medical history, to predict a response of a patient with cancer, such as but not limited to breast cancer, to one or more anti-cancer agents.

[000109] Detection of biomarker to detect cancer, as well as detection of biomarkers for typing and staging of cancer, as well as detection of biomarkers to indicate prognosis of a disease, or the response of a disease to particular therapies, can be by detection of one or more biomarkers underexpressed by cancer cells with respect to normal cells, by detection of one or more biomarkers overexpressed by cancer cells with respect to normal cells, or by a combination of the two. For example, detection of biomarkers can be detection of the proteins of Table 1 or Table 2 identified as upregulated proteins. In addition or in the alternative, detection of biomarkers can be detection of the proteins of Table 1 or Table 2 identified as downregulated proteins.

Antibodies to Biomarkers

[000110] The invention also includes antibodies to biomarkers identified using the methods disclosed herein, including the proteins listed in Table 1 and Table 2. Methods of generating antibodies to proteins are well known in the art. For example, polyclonal antibodies may be isolated and purified from vaccinated animals using procedures well-known in the art (for example, see Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988).

[000111] Antibodies of the invention can also be monoclonal antibodies generated against proteins identified using the methods disclosed herein, such as the proteins listed in Table 1 and Table 2. For example, monoclonal antibodies can be produced following the procedure of Kohler and Milstein (Nature 256:495-497 (1975) (for example, see Harlow et al., supra). Briefly, monoclonal antibodies can be produced by immunizing mice with a biomarker protein, such as a protein of Table 1 or Table 2, verifying the presence of antibody production by removing a serum sample and testing for reactivity against the biomarker protein, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce anti-biomarker antibody, culturing the anti-biomarker antibody-producing clones, and isolating anti-biomarker antibodies from the hybridoma cultures.

[000112] Antibodies of the invention can also be antibody fragments that specifically bind the proteins identified using the methods disclosed herein, such as the proteins listed in Table 1 and Table 2.

[000113] Antibodies of the invention also include engineered antibodies, including, without limitation, humanized antibodies, single-chain antibodies, and recombinant antibodies optimized through phage display. In some embodiments antibodies or antibody fragments can be isolated from antibody phage libraries generated, for example using the techniques described in McCafferty et al. (1990) Nature 348: 552-554, using the antigen of interest (such as a biomarker identified by the methods provided herein) to select for a suitable antibody fragment. Clackson et al. (1991) 352: 624-628 and Marks et al. (1991) J. MoI. Biol. 22: 581-597 describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nanomolar range) human antibodies by chain shuffling (Mark et al. (1992) Bio Technol. 10: 779-783), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al. (1993) Nuc. Acids Res. 21 : 2265-2266). The invention includes bacterial lines comprising phage libraries and phage clones of antibodies generated against biomarkers of the invention, such as the biomarkers listed in Table 1 and Table 2. The invention includes eukaryotic and bacterial lines comprising nucleic acid constructs encoding antibodies generated against biomarkers of the invention, such as the biomarkers listed in Table 1 and Table 2.

[000114] The invention encompasses antibodies that specifically bind the proteins of Table 1 and Table 2, and hybridoma cell lines, bacterial cell lines, and phage that produce antibodies that specifically bind the proteins of Table 1 and Table 2. The invention also encompasses nucleic acid constructs that encode antibodies that specifically bind proteins of Table 1 and Table 2.

[000115] The antibodies of the invention can be used in detection of biomarkers, including but not limited to relative or absolute quantitation of biomarkers. The invention includes methods of detecting expression of a biomarker of Table 1 or Table 2 in a cancer cell or in tissue or bodily fluid removed from a subject known to have, or suspected of having, a neoplasm, cancer, precancerous lesion, premalignant cells, or cells displaying an atypia, using antibodies or other specific binding partners that specifically bind the biomarker, in which the biomarker is detected at a higher or lower level in a cancer cell or in tissue or bodily fluid of a subject known or suspected of having cancerous, precancerous, or premalignant cells, than in normal cells of the same type, or tissues or fluid samples of patients in whom no cancerous or precancerous state has been identified or suspected. The method can include detecting the relative or absolute level of one or more biomarkers of Table 1 and Table 2. Detection of abnormal levels of a biomarker can be correlated with other indicators of a disease or pre-disease state. Other indicators can include, for example, family history, the subjects history of dietary habits or exposure to carcinogens, age, symptoms, cell morphology, mammogram results, sonogram results, levels of other proteins, biomolecules, or metabolites diagnostic of cancer or neoplasms, etc. Results of biomarker expression tests can, together with other data such as these, aid in the diagnosis of a disease state (such as diagnosis of cancer), cancer typing, cancer staging, or prognosis.

[000116] Detection of biomarkers can be by assays of any type, including immunoassays of any type, which can be performed in solution phase (for example, ELISA protocols) or on a substrate, such as a membrane, slide, or bead. Immunoassays are well know in the art. Various methods of generating antibodies and methods for immunoassays are disclosed, for example, in U.S. Patent 6,828,110; U.S. Patent 6,828,110; U.S. Patent 6,828,110; U.S. Patent 6,218,109; U.S. Patent 5,849,508; and U.S. Patent 5,693,778; all herein incorporated by reference in their entireties.

[000117] The present invention also includes methods of detecting biomarker expression by detecting nucleic acids encoding biomarkers, such as the biomarkers of Table 1 and Table 2, in cells or tissue or bodily fluid samples of a subject. Such detection can be, for example, by Northern blot, microarray hybridization, RT-PCR or other polymerase-based assays, CISH, or FISH. The invention includes methods of detecting a nucleic acid encoding at least a portion of a biomarker of Table 1 or Table 2 in a cancer cell or in tissue or bodily fluid removed from a subject known to have, or suspected of having, a neoplasm, cancer, precancerous lesion, premalignant cells, or cells displaying an atypia, in which the biomarker-encoding nucleic acid is detected at a higher or lower level in a cancer cell or in tissue or bodily fluid of a subject known or suspected of having cancerous, precancerous, or premalignant cells, than in normal cells of the same type, or tissues or fluid samples of patients in whom no cancerous or precancerous state has been identified or suspected. The method can include detecting the relative or absolute level of nucleic acids encoding at least a portion of one or more biomarkers of Table 1 and Table 2. Detection of abnormal levels of a biomarker-encoding nucleic acid can be correlated with other indicators of a disease or pre-disease state. Other indicators can include, for example, family history, the subjects history of dietary habits or exposure to carcinogens, age, symptoms, cell morphology, mammogram results, sonogram results, levels of other proteins, biomolecules, or metabolites diagnostic of cancer or neoplasms, etc. Results of biomarker expression tests can, together with other data such as these, aid in the diagnosis of a disease state (such as diagnosis of cancer), cancer typing, cancer staging, monitoring disease progression and/or the efficacy of a particular therapeutic regimen, or prognosis.

Diagnosis of Disease Using Biomarkers

[000118] The abundance of one or more biomarkers in a biological sample can be correlated with a disease state, such as cancer. An altered expression pattern of one or more biomarkers in a biological sample can be correlated with the correlated with disease, such as cancer. The altered expression pattern can be, as nonlimiting examples, a different subcellular localization or post-translational modification.

[000119] One or more biomarkers of a disease state identified using the methods of the present invention can be detected in a plurality of biological samples in which cells of the tissue have been confirmed as having a particular disease state. Preferably, the abundance or subcellular localization of the biomarkers in disease tissue is evaluated at the same times as the abundance of the biomarkers in biological samples of non-disease tissue. The abundance of such biomarkers in biological samples taken from breast cancer patients and sample of non-cancerous breast tissue can be determined using any reliable detection methods, including those disclosed herein. Statistical analysis can be performed to determine correlates of the abundance of particular biomarkers with the disease state.

Correlation of Biomarkers with disease type, stage, prognosis, and response to therapy

[000120] The abundance of one or more biomarkers in a biological sample can be correlated with a type, stage, or prognosis of a disease that the biomarker is indicative of. The abundance of a biomarker in a biological sample can also be correlated with response of the disease to particular therapies, such as drugs. One or more biomarkers of a disease state identified using the methods of the present invention can be detected in plurality of biological samples in which cells of the tissue have been confirmed as having a particular disease state, and have been classified according to one or more of disease type, disease stage, disease prognosis, or response of the patient to a given treatment. Preferably, the abundance of the biomarkers in disease tissue is evaluated at the same times as the abundance of the biomarkers in biological samples of non- disease tissue. Statistical analysis can be performed to determine correlates of the abundance of particular biomarkers with these parameters.

[000121] For example, as described in the examples, the methods of the present invention have been used to identify breast cancer biomarkers, as disclosed in Tables 1 and 2, and provided in the appendix listing of sequences, incorporated by reference herein. The abundance of such biomarkers in biological samples taken from breast cancer patients can be determined using any reliable detection methods, including those disclosed herein. The National Cancer Institute maintains the Cooperative Breast Cancer Tissue Resource (www.cbctr.nci.nih.gov)to supply researchers with primary breast cancer tissues and associated clinical data. Analysis of expression of breast cancer biomarkers, such as those disclosed herein, can be examined in tissues of this tissue bank, for example, and correlated with pathological and clinical information that is available. Such correlates can be used for diagnosing, typing, and staging of cancer using biomarker detection on biological samples of patients known to have or suspected of having cancer. Such correlates can also be used for determining the probability of response of the patient to anticancer agents and a prognosis.

Exemplary biomarkers

[000122] Illustrative biomarkers of present invention include those whose expression levels is discovered to differ by at least about two-fold between breast cancer cells and normal breast cells. Such biomarkers are listed in Table 1 (membrane proteins) and in Table 2 (secreted proteins). The sequences of these proteins are provided in attached appendices, which are hereby incorporated into the specification by reference.

[000123] Some exemplary biomarkers of Table 1 that can be used in the methods of the invention include periostin, osteoblast specific factor, tyrosine kinase receptor, roundabout 1 , and hydroxymethylglutaryl-CoA lyase. Some exemplary biomarkers of Table 2 that can be used in the methods of the invention include the interferon inducible double stranded RNA dependent protein kinase (gi number 4506103) and platelet proteoglycan (gi 16197601). [000124] The proteins whose differential expression by breast cancer and normal breast cells was demonstrated by the methods of the invention were identified by database searching. Thus the names of the proteins may include "precursor" or "isoform" because these reflect the title of the sequence entries in the sequence database. The biomarkers of the invention are not limited to particular forms of these proteins, however, and encompass all forms of a protein encoded at a particular locus by a particular gene that encodes a protein listed herein. A biomarker of the invention thus includes a protein listed in Table 1 or Table 2 and includes alternative isoforms, processed forms, and post-translationally modified forms of the listed proteins.

[000125] The following examples are intended to illustrate but not to limit the invention.

EXAMPLE 1

IDENTIFICATION OF MEMBRANE PROTEINS DIFFENTIALLY EXPRESSED IN

BREAST CANCER CELLS

[000126] Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and accurate approach to quantify differential protein expression and dynamic regulation of posttranslational modification. Two populations of cells are grown in identical media except that one contains light amino acids and the other contains heavy amino acids. For example, ^Relabeled amino acids, such as [U-¹³C₆]LyS and [U-¹³Ce]Arg, are stable isotopes and can be handled like regular amino acids. During cell culturing, light or heavy amino acids are incorporated into proteins using the natural biosynthetic machinery of the cells. The labeling efficiency is almost 100%. Cells labeled with light or heavy amino acids are combined and treated as a single sample prior to any process or protein purification, eliminating quantification error due to unequal sample preparation and increasing reproducibility. When dual labels of heavy Lys and Arg are used, all tryptic peptides carry a mass tag with exception of C-terminal peptides, because trypsin cleaves polypeptides after Lys or Arg. Proteins and peptides labeled with light or heavy amino acids are chemically identical and co-elute in any liquid chromatography or electrophoretic separations. Nevertheless, the mass difference between the light and heavy peptides was distinguishable by mass spectrometry. Once isotopic peptide pairs have been correlated with the proteins from which they originate, their relative intensities can be used to quantify different expression of the parent protein between normal and disease state cells.

[000127] Membrane proteins play a pivotal role in regulating cell-cell interaction, recognition, migration, adhesion, and signal transduction. Currently more than 50% of all major drug targets for medicines are membrane proteins. In this report, we describe a SILAC approach in the quantification of differential membrane expression between normal and malignant breast cells from cell lines derived from a 74-year-old female with breast carcinoma.

Materials

[000128] NuP AGE® gels, NuP AGE® sample buffer, SimplyBlue SafeStain, Invitromass LMW calibrants, DMEM medium, Lys and Arg-deprived DMEM medium, FBS, dialyzed FBS, epidermal growth factor (EGF), SuperPicTure™ Polymer Detection Kit, and monoclonal anti- CD 13 antibody were obtained from Invitrogen Life Sciences (Carlsbad, CA). [U-¹³Ce] L-Lysine and [U-¹³Ce₁U-¹⁵N₄] L-Arginine were purchased from Cambridge Isotope Laboratories. Normal (light) L-Lysine, normal (light) L-Arginine, Aprotinin, Leupeptin Hemisulfate, phenylmethanesulfonyl fluoride (PMSF), and insulin were purchased from Sigma. Rabbit anti- osteoblast specific factor 2 (OSF-2) antibody was ordered from Biovendor Laboratory Medicine, Inc. Trypsin was ordered from Promega. Benzonase was from Novagen. Dynabeads®- streptavidin were from Invitrogen (Carlsbad, CA), and sulfo-NHS-SS-biotin was obtained from Pierce. Normal (HTB- 125) and malignant (HTB- 126) breast cells, isolated from a 74 female with breast carcinoma, were purchased from ATCC.

Isolation of Membrane Surface Proteins or integral membrane proteins

[000129] Normal breast cells were maintained in DMEM medium containing 10% FBS and 30 ng/ml EGF and malignant cells were maintained in DMEM medium containing 10% FBS and 30 ng/ml insulin. For labeling of cells with light or heavy amino acids, aliquots of normal and malignant breast cells were harvested separately. Normal breast cells were resuspended in 3 ml of modified DMEM medium supplemented with 10% dialyzed FBS, 30 ng/ml EGF, light L- Lysine and light L-Arginine, whereas malignant cells were resuspended in 3 ml of modified DMEM medium supplemented with 10% dialyzed FBS, 30 ng/ml insulin, heavy [U-¹³Ce] L- Lysine and heavy [U-¹³Ce₁U-¹⁵N₄] L-Arginine. Initially normal and malignant cells were cultured in two separate 60 mm dishes. Every three to four days, the cells were split or the media replaced with the corresponding light or heavy labeling medium. It takes six doubling times to achieve almost 100% incorporation of heavy amino acids into proteins, which means the total cell number would be 64 x 10⁵ when starting with 1 x 10⁵ cells. It is advisable to normalize the number of normal and malignant cells by cell counting before cell surface labeling and mixing. For cell surface biotinylation, two plates (~10⁶ cells/100 mm dish) each of normal and malignant breast cells were washed twice with PBS and then incubated with 5 ml of PBS containing 0.25 mg/ml of sulfo-NHS-SS-biotin. The plates were gently agitated at 4 ⁰C for 30 minutes and then the reaction was quenched with 0.5 ml of TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 200 mM Glycine. Normal and malignant breast cells were scraped off the dishes and mixed at 1 : 1 ratio. Cells were harvested by centrifugation at 1000 rpm for 10 minutes.

[000130] For isolation of cell surface membrane proteins, cell pellets were lysed in 2 ml of detergent-containing lysis buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5% CHAPS, and protease inhibitor cocktails) using 50 passes of a dounce homogenizer. The cell lysate was centrifuged at 100,000 x g for 20 minutes and the supernatant was incubated with 1 mg of Dynabeads MyOne streptavidin for 1 h at 4⁰C with gentle rotation. The beads were captured to the side of the tube using a magnet and washed three times with lysis buffer before being resuspended in 40 μl of 2 x SDS sample buffer containing 50 mM DTT. The mixture was incubated at room temperature for 30 minutes with gentle vortex before being recaptured to the side of the tube. The supernatant was transferred to a new tube, heated at 95 ⁰C for 5 minutes, and loaded on a SDS-PAGE.

[000131] For isolation of integral membrane proteins, cell pellets were lysed on ice for 30 min in 1.6 ml of hypotonic buffer (10 mM Tris-HCl (pH 7.4), 1 mM MgCl₂, 0.5 mM PMSF, 0.15 μM Aprotinin, 1 μM Leupeptin Hemisulfate, and 10 U/ml benzonase), followed by 30 strokes of a Dounce homogenizer. To the cell suspension, 0.4 ml of 5 x sucrose (1.25 M sucrose stock in H₂O) was added and mixed for five times in the homogenizer, followed by centrifugation at 500 x g for 10 min to remove nuclei. 1 mg of Dynabeads Myone streptavidin was added to the supernatant and the mixture was gently rotated for 1 h at 4 ⁰C. The beads were captured using a magnet and washed three times with 1 ml of hypotonic buffer, three times with 1 mL of 1 M KCl, and three times with 1 mL of 0.1 M Na₂CO₃, respectively. Finally the beads were resuspended in 50 μl of 2 x SDS sample buffer containing 50 mM DTT. The mixture was vortexed at room temperature for 30 minutes. The beads were recaptured and the supernatant was transferred to a new tube, heated at 95 ⁰C for 10 minutes, and loaded on a SDS-PAGE.

[000132] The entire gel lane was cut into as many as 45 fractions and the gel pieces were subjected to in-gel tryptic digest. Tryptic peptides of cell surface and plasma membrane proteins labeled with light or heavy amino acids were analyzed with nanoelectrospray LC-MS/MS on a Q-TOF API-US instrument (Waters Corporation). Atlantis™ dC18, 3 μm, 100 μm x 100 mm column (Waters Corporation) was used for peptide separation. Alternatively, peptides were fractionated and spotted onto MALDI plates in a Nano LC-Probot system (LC Packings, Dionex), followed by analysis with a 4700 proteomics analyzer (Applied Biosystems). For simple digests, a gradient of 5-45% (v/v) acetonitrile in 0.1% formic acid over 45 min, and then 45-95% acetonitrile in 0.1% formic acid over 5 min was used. For a complex sample, a gradient of 5-45% (v/v) acetonitrile over 90 min, and then 45-95% acetonitrile over 30 min was used. On the Q-TOF, four components were used to acquire MS/MS data with 1.4 s scan time. On the 4700 proteomics, peptides with mass over 1000 were chosen for MS/MS analysis.

[000133] Raw data files from Q-TOF instrument were processed with Mascot Distiller (Matrix Science, London) and then searched against NCBI database using Mascot search algorithm. The Mascot search result can show identities of proteins as well as the relative ratio of isotopic peptide pairs.

[000134] Figure 2 demonstrates that using these methods, the quantitation of different peptides originating from a single membrane protein (platelet derived growth factor receptor) that was not present at different levels in normal (L, light isotope) and cancer (H, heavy isotope) cells was remarkably consistent. Figure 3 demonstrates that quantitation of a membrane protein in three separate experiments was also consistent. Figure 4 provides examples of mass spectra of peptides derived from proteins determined to be upregulated and downregulated on the membranes of breast cancer cells when compared with normal breast cells.

[000135] Table I provides proteins identified as having a different abundance in breast cancer cells versus normal cells using two different methods for membrane protein isolation. Using these methods, we have identified and quantified over 300 cell surface proteins and 500 plasma membrane proteins. A majority of proteins remained unchanged between normal and malignant cells. However, as seen in Table 1, some matrix proteins and ion channel proteins for salts and amino acids, show increased expression levels in malignant breast cells, whereas some metalloproteases, disulfide-isomerases, cis-trans isomerases exhibited decreased expression levels in malignant cells. These results indicate that SILAC is a powerful technique for global identification and quantification of cell surface proteins and plasma membrane proteins between normal and diseased states. Normal and malignant breast cells, isolated from the same patient with primary breast cancer, were metabolically labeled with light and heavy Lysine and Arginine, respectively, as described in Example 1.

Table I: Plasma Membrane Proteins Differentially Expressed in Breast Cancer Cells

NBCI Gl SILAC

UPREGULATED PROTEINS NUMBER RATIO

SEQ ID

5453834 10.2 periostin, osteoblast specific factor; NO:1

SEQ ID

4505591 -3.2 peroxiredoxin 1 ; NO:2

SEQ ID farnesyl-diphosphate M 435677 3.9

INfVJ)-.Q O farnesyltransferase

SEQ lD tyrosine kinase receptor NO:4 292870 10.5

SEQ ID epidermal growth factor receptor Mfi-c; 66820 8.7

(precursor)

SEQ lD 480007 5.8 osteoblast-specific factor 2 NO:6

SEQ ID NO:7 18204869 3.3

K-ALPHA-1 protein

SEQ ID 4506569 9.4 roundabout 1 isoform a; roundabout NO:8 1 ; axon guidance receptor

RD114/simian type D retrovirus SEQ ID

4191556 6.5 receptor NO:9

SEQ ID unnamed protein product NO:10 22760207 5.5 alpha 1 type I collagen preproprotein; SEQ ID

Collagen I, alpha-1 polypeptide; NO:11 4502945 3 SEQ ID amino acid transporter E16 NO:12 3639058 5.1 SEQ ID 4F2 antigen heavy chain NO:13 177207 4.1 SEQ ID unnamed protein product NO:14 36102 3.8

Ephrin type-A receptor 2 precursor

SEQ ID (Tyrosine-protein kinase receptor 125333 3.5 NO:15 ECK)

Solute carrier family 7 (cationic

SEQ ID amino acid transporter, y+ system), NO:16 27503713 4.1 member 5

SEQ ID unnamed protein product NO:17 34530952 >12 SEQ ID neutral amino acid transporter B NO:18 1478281 5.6 SEQ ID pancreatic tumor-related protein NO:19 189597 3.9

NBCI Gl SILAC

DOWNREGULATED PROTEINS NUMBER RATIO

SEQ ID autoantigen, 64K NO:20 284006 3.5 SEQ ID integrin alpha 2 [Homo sapiens] NO:21 3764061 SEQ ID P63 protein NO:22 479303 4.2

SEQ ID membrane alanine aminopeptidase NO:23 4502095 3.5 precursor

SEQ ID

FK506 binding protein 9 (Peptidyl- NO:24 23396584 6.5 prolyl cis-trans isomerase) (PPIase)

SEQ ID thioredoxin peroxidase NO:25 5453549 -3.2 SEQ ID reticulocalbin 1 precursor NO:26 4506455 5.5 SEQ ID heme oxygenase (decyclizing) 1 NO:27 4504437 4.9 SEQ ID GTP-binding protein Rab3B NO:28 106187 -6.9 Hydroxyacyl-Coenzyme A SEQ ID

12653209 4.3 dehydrogenase, type Il NO:29 hydroxysteroid dehydrogenase-like SEQ ID protein 33694276 4.1

NO:30

SEQ ID

7643782 3.4

HDCMD47P NO:31

SEQ ID hydroxymethylglutaryl-CoA lyase NO:32 184503 7.8

SEQ ID protein disulphide isomerase, PDI

INVJ. OO 254299

{N-terminal}

SEQ lD protein disulfide-isomerase (EC M QA 1085373 3.2

WfKJI-.OH

5.3.4.1 ) ER60 precursor

SEQ ID

Type I inositol-1 ,4,5-trisphosphate 5- 3122245 5.2 phosphatase (5PTASE)

SEQ ID unnamed protein product 28678 4.5

NO: 36

SEQ ID

177872 5.1 alpha-2-macroglobulin NO:37

SEQ ID

19743813 4.3 integrin beta 1 isoform 1A precursor NO: 38

SEQ ID 2809324 5.8 calumenin NO: 39

SEQ ID

4929557 7.6

CGI-44 protein NO:40

SEQ ID

3 endocytic receptor Endo180 NO:41 4835878

SEQ ID

17225574 4.1

LIM domain only 7 NO: 42

Aspartate aminotransferase SEQ ID

2 precursor 4504069 4.2

NO:43

Cysteine-rich fibroblast growth factor SEQ ID

1373019 3.1 receptor NO:44

SEQ ID phospholipase C-alpha 303618 3.5

NO:45

Human protein disulfide isomerase, SEQ ID

Nmr 2098329 3.7

NO:46

SEQ ID

Progesterone membrane binding 16876814 NO:47 protein

Protein disulfide-isomerase A3 SEQ ID precursor (Disulfide isomerase ER- 729433 3.4

NO:48

60) (ERP60)

SEQ ID unnamed protein product 35655 4.4 NO:49

SEQ ID lamin A protein 386856 -3.3

NO:50

SEQ ID

KIAA2019 protein 24899202 8.5 NO:51

Lvsosome-associated membrane SEQ ID 126376 4.3 protein-3 variant NO: 52

SEQ ID ubiquitin activating enzyme E1 35830 4.3

NO:53

SEQ ID

Pregnancy zone protein precursor 131756

NO:54 8.3

Human basement membrane heparin SEQ ID

29470 Sulfate proteoglycan core protein 5.1

NO: 55

SEQ ID

34228 unnamed protein product 3

NO:56

SEQ ID mitochondrial processing peptidase beta- 3342006 3.8 NO:57 subunit

SEQ ID mitochondrial dihydrolipoamide 499719 NO:58 succinyltransferase

SEQ ID

Lysosome-associated membrane 21070332 4.2 NO:59 glycoprotein 1 precursor (LAMP-1 ) Progesterone receptor membrane SEQ ID

5729875 4.2 component 1 NO:60 SEQ ID

339647 5.7 thyroid hormone binding protein precursor NO:61

SEQ ID

Peptidyl-prolyl cis-trans isomerase B 118090 NO:62 3 precursor (PPIase) (Rotamase)

SEQ ID

Sideroflexin 3 20140223 3.2 NO:63

EXAMPLE 2

IDENTIFICATION OF SECRETED PROTEINS DIFFENTIALLY EXPRESSED IN

BREAST CANCER CELLS

[000136] Normal and malignant breast cells, isolated from the same patient with primary breast cancer, were metabolically labeled with light and heavy Lysine and Arginine, respectively, as described in Example 1. Upon reaching 80% confluence, cells were washed and incubated in serum-free media for 48 h. The Light and heavy-labeled media from the cultures were harvested and centrifuged, then mixed at 1 : 1 ratio and concentrated using a MW 5000 Da cut off Amicon ultrafiltration membrane. Samples were analyzed by SDS-PAGE on 4-20% Novex gels followed by in-gel tryptic digest. Extracted peptides were analyzed using capillary LC-MS/MS and proteins were assigned using Mascot Server. The procedure for secreted biomarker identification is summarized in Figure 5. Relative quantification of secreted proteins was based on the ratio of isotopic peptide pairs in the MS spectrum. Potential biomarkers were then validated using western blotting or ELISA. [000137] Using these methods, over 400 secreted proteins were identified and quantified in these experiments. Figure 6 illustrates the consistency in quanitation of by mass spectra of four peptides of in these experiments. Figure 7 demonstrates the experiment-to-experiment variation in peptide quantitation in these procedures is negligible.

[000138] The majority of the isolated and identified proteins were matrix proteins, cytoskeleton proteins, and proteins involved in metabolism and signal transduction. More than 10% of the secreted proteins were proteases or protease inhibitors and approximately 4.5% of the secreted proteins were growth factors, cytokines, and chemokines. As seen in Figure 8, OSF-2, a protein having increased abundance in the media of breast cancer cells (C) relative to the media of normal breast cells (N) as determined by SILAC and mass spectrometry (Figure 8A) is also demonstrated to have reduced abundance in the media of breast cancer cells when tested by Western blot. Similarly, serpinE2, a protein found by SILAC/mass spectrometry to have reduced abundance in the media of breast cancer cells (C) with respect to the media of normal breast cells (N) is also validated as a biomarker by Western blot (Figure 8B).

[000139] The majority of proteins did not show different abundance between normal and malignant breast cells. As shown in Table 2, a number of matrix proteins and protease inhibitors did show decreased expression levels in malignant cells, suggesting that down regulation of protease inhibitors is essential for tumor cell migration and invasion. On the other hand, some matrix proteins, such as osteoblast-specific factor 2, exhibited increased expression in malignant cells. Differential expression of some targeted proteins has been validated using Western Blotting (Figure 8B) or ELISA. These results indicated that SILAC is a powerful technique for initial identification and quantification of secreted proteins that correlate with tumor invasiveness.

[000140] Western blot of normal breast tissue and breast cancer cells were found to have altered expression in breast cancer cells when compared with normal breast cells. Thus, the detection of biomarkers using SILAC is reliable and verifiable using other methods for comparison of expression levels or patterns.

Table II: Secreted Proteins Differentially Expressed by Breast Cancer Cells NBCI Gl SILAC

UPREGULATED PROTEINS NUMBER RATIO

SEQ ID alpha 1 type 1 collagen preprotein 4502945 5 ± 0.6 NO:11 protein kinase, interferon-inducible SEQ ID 4506103

7.9 double stranded RNA dependent NO:64 alpha 2(1 ) collagen SEQ ID 2388555

3.8 ± 0.4 NO:65 neuropeptide NPVF precursor SEQ ID 15281404 9.1

NO:66 skeletal muscle LIM-protein SLIM SEQ ID 1381814 4.1

NO: 67 extracellular matrix protein periostin- SEQ ID 23345100 16.5 bm NO: 68 periostin, osteoblast specific factor SEQ ID 5453834 10.3

NO: 1 osteoblast-specific factor 2 SEQ ID 480007 12.1

NO: 6 Platelet proteoglycan (125 AA) SEQ ID 55666241 20 ± 3.0

NO: 69

SEQ ID type III preprocollagen alpha 1 chain 16197601 3.2 ± 0.4 NO: 70

NBCI Gl SILAC

DOWNREGULATED PROTEINS

NUMBER RATIO

PAI precursor polypeptide SEQ ID 31147 3

NO:71 peroxiredoxin 1 SEQ ID 4505591 3

NO: 72 SERPINE2 protein SEQ ID 27769056 11.7

NO: 73 antithrombin III SEQ ID 179161 3.7

NO: 74 pigment epithelial-differentiating SEQ ID 423038 6.8 factor precursor NO: 75 perlecan precursor - human SEQ ID 539611 3.8

NO: 76 alpha-2-macroglobulin SEQ ID 177872 12.4

NO: 37 amyloid beta A4 precursor protein- SEQ ID binding, family B, member 1 isoform NO:77 4502131 3.5 E9 decorin SEQ ID

181519 4.7 NO:78

Annexin A2 SEQ ID 16306978 3.7

NO:79 fibronectin precursor SEQ ID 31397 3.1

NO:80 Annexin 5 SEQ ID 17391477 4.3

NO:81 insulin-like growth factor binding SEQ ID 183894 4 protein 6 NO:82 SEQ ID macroglobulin alpha2 224053 10.2 NO:83 Pregnancy zone protein precursor SEQ ID 131756 9.1 NO: 84 plasma protease (C 1 ) inhibitor SEQ ID 179619 13 precursor NO:85

Inter-alpha-trypsin inhibitor heavy 125000 9.4

SEQ ID chain H2 precursor (ITI heavy chain NO:86

H2) pregnancy-specific beta-1 SEQ ID 551604 7.7 glycoprotein NO:87

ISLR SEQ ID 37182860 4.5

NO:88 laminin A3 SEQ ID 509806 18 NO: 89 von Ebner minor protein SEQ ID 19880274 6.2

NO:90 Laminin alpha 4 chain SEQ ID 1042082 5.4 ±

NO:91 0.6 unnamed protein product SEQ ID 31189 4.2

NO:92 tissue inhibitor of metalloproteinases- SEQ ID 1517893 3

2 NO:93

TIMP-2, CSC-21 K=tissue inhibitor of SEQ ID 262883 metalloproteinase NO:94

Pregnancy specific beta-1 - SEQ ID 38649024 glycoprotein 4, isoform 1 5 NO:95 antithrombin-TRI, AT-TRI SEQ ID 998404 10.5

{internal fragment} NO:96 exon 3a SEQ ID 457132 11.2

NO:97

S-laminin SEQ ID 288401 5.6

NO:98

Human Annexin V With Proline SEQ ID Substitution By Thioproline NO:99 3212603 4.1 plasminogen activator inhibitor 1 SEQ ID 189578 10.5 NO:100 plasminogen activator inhibitor type SEQ ID 24307907 11.2

1 , member 2 NO:101 similar to Pacific ray VAT1 protein, SEQ ID 602278 3.1 NO:102

Pregnancy specific beta-1 - SEQ ID 15214951 8.9 glycoprotein 5 NO:103 precursor polypeptide (AA -31 to SEQ ID 37465 4.5

1139) NO:104 hypothetical protein SEQ ID 34365344 10.2 NO:105

Human basement membrane heparin SEQ ID 29470 4 ± 0.5 sulfate proteoglycan core protein NO:106 Insulin-like growth factor binding SEQ ID 12652547 3.6 ± protein 3 NO:107 0.4

Matrix metalloproteinase 3 preprotein SEQ ID 4505217 16 ± 2

NO:108

Prostaglandin D2 synthase 21 kD SEQ ID 54696706 6.4 ±

NO:109 0.7

Tumor necrosis factor SEQ ID 339992 4 ± 0.5

NO:110 thrombospondin SEQ ID 538354 3.2 ±

NO:110 0.4 collagenase type IV precursor SEQ ID 180671 10.3

NO:112

EXAMPLE 3

IDENTIFICATION OF MICRO RNAS DIFFENTIALLY EXPRESSED IN BREAST

CANCER CELLS

[000141] Normal (HTB-125) and malignant (HTB-126) breast cells, isolated from the same patient with primary breast cancer, were grown as in Example 1, but in the absence of heavy isotope, and three individual batches of RNA were isolated from harvested cells using a PURELINK™ miRNA isolation kit, as described by the manufacturer (Invitrogen, Carlsbad, CA) . miRNA was labeled using NCODE™ miRNA Labeling System and hybridized to an NCODE™ Multi-Species miRNA Microarray according to manufacturers instructions.

[000142] Data from the array hybridization were background corrected, normalized and averaged. Proteins of the same cell types were analyzed via SILAC and mass spectrometry. The heat map on the left panel demonstrates the change in miRNA and protein levels between normal and malignant breast cell types, where up-regulation is indicated in red and down-regulation in green. Detailed information and seed region alignment for a subset of the data is shown in the right panel.

[000143] Microarray data was verified using the NCODE™ SYBR® Green miRNA qRT-PCR system. qRT-PCR was performed on 8 of the microRNAs that showed a significant fold change on the microarray. The qRT-PCR results correlated with the microarray data, as exhibited by fold change values in Figure 9. [000144] miR Iet7d (AGAGGUAGUAGGUUGCAUAGU, SEQ ID NO:112), miR 17-5p (CAAAGUGCUUACAGUGCAGGUAGU, SEQ ID NO:113);

miR 20-a (UAAAGUGCUUAUAGUGCAGGUAG, SEQ ID NO:114);

miR 21 (UAGCUUAUCAGACUGAUGUUGA, SEQ ID NO:115);

miR-30b (UGUAAACAUCCUACACUCAGCU, SEQ ID NO:116);

miR-106a (AAAAGUGCUUACAGUGCAGGUAGC, SEQ ID NO:117);

miR-106b (UAAAGUGCUGACAGUGCAGAU, SEQ ID NO:118);

miR- 195 (UAGCAGCACAGAAAUAUUGGC, SEQ ID NO: 119) showed from an approximately one-fold to an approximately six-fold increase in malignant breast cells with respect to normal breast cells Figure 9.

[000145] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention.

[000146] All headings are for the convenience of the reader, and are not intended to limit the scope of the invention. All references cited herein, including patents, patent applications, and publications, are incorporated by reference in their entireties.

Claims

CLAIMS What is claimed is:

1. A method comprising: contacting a sample with a specific binding member, wherein the specific binding member specifically binds to a protein listed in Table 1 or Table 2; and detecting binding of the specific binding member to a protein listed in Table 1 or Table 2 in the sample, wherein the sample is a sample from a subject known to have or at risk of or suspected of having breast cancer, a breast neoplasm, or a precancerous breast lesion.

2. The method of claim 1, further comprising determining the relative or absolute amount of the protein listed in Table 1 or Table 2.

3. A method of detecting one or more biomarkers expressed by a cancer cell, comprising:

providing a biological sample comprising cancer cells or biomolecules derived from cancer cells from a subject with cancer or at risk of or suspected of having cancer, a neoplasm, a precancerous lesion, or premalignant cells;

detecting one or more biomarkers of Table 1 or Table 2, or one or more nucleic acid molecules encoding one or more biomarkers of Table 1 or Table 2 in the biological sample.

4. The method of claim 1 or 3, wherein said biological sample is a tumor biopsy sample.

5. The method of claim 1 or 3, wherein said biological sample comprises blood, plasma, serum, urine, cerebrospinal fluid, lymphatic fluid, pelvic lavage, lung aspirate, nipple aspirate, or breast duct lavage.

6. The method of claim 1 or 3, wherein said one or more biomarkers is two or more biomarkers of Table 1 and/or Table 2.

7. The method of claim 1 or 3, wherein said one or more biomarker is three or more biomarkers of Table 1 and/or Table 2.

8. The method of claim 1 or 3, wherein said detecting one or more biomarkers identified by the method of claim 1 is by immunocytochemistry, ELISA, Western, antibody array, affinity capture, mass spectrometry, or functional assay.

9. The method of claim 1 or 3, wherein said detecting one or more nucleic acid molecules encoding one or more biomarkers is by Northern blot, RT-PCR, nucleic acid microarray hybridization, or FISH.

10. The method of claim 1 or 3, further comprising correlating the detection of said one or more biomarkers with a type of cancer.

11. The method of claim 1 or 3, further comprising correlating the detection of said one or more biomarkers with a stage of cancer.

12. The method of claim 1 or 3, further comprising correlating the detection of said one or more biomarkers with a prognosis.

13. The method of claim 1 or 3, further comprising correlating the detection of said one or more biomarkers with response to one or more anti-cancer therapies, particularly treatment with one or more anti-cancer agents.

14. The method of claim 1 or 3, wherein an altered expression level in the biological sample compared to a normal sample is indicative of breast cancer.

15. A method of detecting one or more biomolecules, comprising detecting in a biological sample, expression of a protein of Table 1 or Table 2, or a nucleic acid encoding a protein of Table 1 , wherein said biological sample is a sample of a patient with a breast pathology.

16. The method of claim 15, wherein said biological sample is a tumor biopsy sample.

17. The method of claim 15, wherein said biological sample is a breast tumor biopsy sample.

18. The method of claim 15, wherein the biological sample is a serum sample.

19. The method of claim 15, wherein said biological sample comprises blood, plasma, serum, urine, saliva, cerebrospinal fluid, lymphatic fluid, pelvic lavage, lung aspirate, nipple aspirate, or breast duct lavage.

20. The method of claim 15, wherein expression is detected for two or more proteins of Table 1 and/or Table 2, or nucleic acids encoding two or more proteins of Table 1 and/or Table 2.

21. The method of claim 15, wherein expression is detected for three or more proteins of Table 1 and/or Table 2, or nucleic acids encoding three or more proteins of Table 1 and/or Table 2.

22. The method of claim 15, wherein expression is detected for four or more proteins of Table 1 and/or Table 2, or nucleic acids encoding four or more proteins of Table 1 and/or Table 2.

23. The method of claim 15, wherein an altered expression level in the biological sample compared to a normal sample is indicative of the presence of a breast pathology.

24. The method of claim 15, wherein an altered expression level in the biological sample compared to a normal sample is indicative of the presence of breast cancer.

25. The method of claim 15, further comprising correlating the expression level of the protein or the nucleic acid with a type of cancer.

26. The method of claim 15, further comprising correlating the expression level of said protein or nucleic acid with a stage of cancer.

27. The method of claim 15, further comprising correlating the expression level of said protein or nucleic acid with a prognosis.

28. The method of claim 15, further comprising correlating the expression level of said protein or nucleic acid with response to one or more anti-cancer agents.

29. The method of claim 15, wherein the detecting comprises detecting binding between a specific binding reagent that binds to the protein or the nucleic acid from the biological sample.

30. The method of claim 29, wherein the specific binding member is an antibody or a nucleic acid.

31. The method of claim 15, wherein the detecting comprises an immunoassay.

32. A kit comprising a specific binding member that binds to a protein of Table 1 or Table 2, or that binds to a nucleic acid encoding a protein of Table 1 or Table 2, and a positive control.

33. The kit of claim 32, wherein the specific binding member is an antibody or a nucleic acid.

34. The kit of claim 32, further comprising a specific binding member that binds to a second protein of Table 1 , or that binds to a nucleic acid encoding a second protein of Table 1 or Table

2.

35. A method comprising : contacting a sample with a specific binding member that binds a micro RNA, wherein the specific binding member specifically binds to miR Iet7d (SEQ ID NO:112), miR 17-5p (SEQ ID NO:113); miR 20-a (SEQ ID NO:114); miR 21 (SEQ ID NO: 115); miR-30b (SEQ ID NO: 116); miR- 106a (SEQ ID NO: 117); miR- 106b (SEQ ID NO: 118); or miR- 195 (SEQ ID NO: 119); and detecting binding of the specific binding member to miR Iet7d (SEQ ID NO:112), miR 17-5p (SEQ ID N0:l 13); miR 20-a (SEQ ID N0:l 14); miR 21 (SEQ ID N0:l 15); miR-30b (SEQ ID NO: 116); miR- 106a (SEQ ID NO: 117); miR- 106b (SEQ ID NO: 118); or miR- 195 (SEQ ID NO: 119) in the sample, wherein the sample is a sample from a subject known to have or at risk of or suspected of having breast cancer, a breast neoplasm, or a precancerous breast lesion.

36. A method of detecting one or more miRNA biomarkers expressed by a cancer cell, comprising:

detecting one or more, or one or more miRNA molecules.

37. The method of claim 35 or 36, wherein said biological sample is a tumor biopsy sample.

38. The method of claim 35 or 36, wherein said biological sample comprises blood, plasma, serum, urine, cerebrospinal fluid, lymphatic fluid, pelvic lavage, lung aspirate, nipple aspirate, or breast duct lavage.

39. The method of claim 35 or 36, wherein one or more miRNAs is miR Iet7d (SEQ ID NO: 112), miR 17-5p (SEQ ID NO: 113); miR 20-a (SEQ ID NO: 114); miR 21 (SEQ ID NO: 115); miR-30b (SEQ ID NO: 116); miR-106a (SEQ ID NO: 117); miR-106b (SEQ ID NO:118); or miR-195 (SEQ ID NO:! 19)