AU2015215980B2 - Insulin receptor substrate 1 (IRS1) protein SRM/MRM assay - Google Patents

Abstract

Abstract The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the Insulin Receptor Substrate 1 (IRS 1 ) protein that are particularly advantageous for quantifying the IRS 1 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein said biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid TissueTM reagents and protocol and the IRS 1 protein is quantitated in the Liquid Tissue TM sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an IRS 1 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

Description

Insulin Receptor Substrate I (IRS!) Protein SRM/MRM Assay 2015215980 24 Aug 2015

This is a divisional application of Australian patent application No. 2010341482, the entire contents of which are incorporated herein by reference.

This application claims the benefit of U.S. Provisional Application 61/289,382, filed December 22, 2009, entitled "Insulin Receptor Substrate 1 (IRSi) Protein SRM Assay" naming as an inventor David B. Krizman, the entirety of which is incorporated by reference.

Introduction

Specific peptides derived from subsequences of the Insulin Receptor Substrate i protein mid which will be referred to as IRSI are provided. The peptide sequence and fragmentation/transition ions for each peptide are particularly useful in a mass spectrometry-based Selected Reaction Monitoring (SRM), which can also be referred to as a Multiple Reaction Monitoring (MRM) assay, and will be referred to as SRM/MRM. Information about the use of peptides for SRM/MRM quantitative analysis of the IRSI protein is described.

This SRM/MRM assay can be used to measure relative or absolute quantitative levels of one or more of the specific peptides from the IRSI protein and therefore provide a means of measuring the amount of the IRSI protein in a given protein preparation obtained from a biological sample by mass spectrometry.

More specifically, the SRM/MRM assay can measure these peptides directly in complex protein lysate samples prepared from cells procured from patient tissue samples, such as formalin fixed cancer patient tissue. Methods of preparing protein samples from formalin fixed tissue are described in U.S. Patent No. 7,473,532, the contents of which are hereby incorporated by references in their entirety. The methods described in U.S. Patent No. 7,473,532 may conveniently be carried out using Liquid Tissue™ reagents and protocol available from Expression Pathology Inc. (Rockville, MD).

The most widely and advantageously available form of tissues from cancer patients tissue is formalin fixed, paraffin embedded tissue. Formaldehyde/formalin fixation of surgically removed tissue is by far and away the most common method of preserving cancer tissue samples worldwide and is the accepted convention for standard pathology practice. Aqueous solutions of formaldehyde are referred to as formalin. ”100%" formalin consists of a saturated solution of formaldehyde (this is about 40% by volume or 37% by mass) in water, with a small amount of stabilizer, usually methanol to limit oxidation and degree of polymerization. The most common way in which tissue is preserved is to soak whole tissue for extended periods of time (8 hours to 48 hours) in aqueous formaldehyde, commonly 1 termed 10% neutral buffered formalin* followed by embedding the fixed whole tissue in paraffin wax for long term storage at room temperature. Thus molecular analytical methods to analyze formalin fixed cancer tissue will be the most accepted and heavily utilized methods for analysis of cancer patient tissue. 2015215980 24 Aug 2015

Results from the SRM/MRM assay can be used to correlate accurate and precise quantitative levels of the IRS1 protein within the specific tissue samples (e.g„ cancer tissue sample) of die patient or subject from whom the tissue (biological sample) was collected and preserved. This not only provides diagnostic information about the cancer, but also permits a physician or other medical professional to determine appropriate therapy for the patient. Such an assay that provides diagnostically and therapeutically important information about levels of protein expression in a diseased tissue or other patient sample is termed a companion diagnostic assay. For example, such an assay can be designed to diagnose the stage or degree of a cancer and determine a therapeutic agent to which a patient is most likely to respond.

Summary

The assays described herein measure relative or absolute levels of specific unmodified peptides from the IRS 1 protein and also can measure absolute or relative levels of specific modified peptides from the IRS! protein. Examples of modifications include phosphorylated amino acid residues and glycosylated amino acid residues that are present on the peptides.

Relative quantitative levels of the IRS 3 protein are determined by the SRM/MRM methodology for example by comparing SRM/MRM signature peak areas (e.g., signature peak area or integrated fragment ion intensity) of an individual IRS 1 peptide in different samples. Alternatively, it is possible to compare multiple SRM/MRM signature peak areas for multiple IRS 1' signature peptides, where each peptide has its own specific SRM/MRM signature peak, to determine the relative IRS1 protein content in one biological sample with the 1RS1 protein content in one or more additional or different biological samples. In this way, the amount of a particular peptide, or peptides, from the IRS 1 protein, and therefore the amount of the IRS1 protein, is determined relative to the same 1RS1 peptide, or peptides, across 2 or more biological samples under the same experimental conditions. In addition, relative quantitation can be determined for a given peptide, or peptides, from the IRS1 protein within a single sample by comparing the signature peak area for that peptide by SRM/MRM 2 methodology to the signature peak area for another and different peptide, or peptides, from a different protein, or proteins, within the same protein preparation from the biological sample. In this way, the amount of a particular peptide from the IRS 1 protein, and therefore the amount of the IRS 1 protein, is determined relative one to another within the same sample. These approaches generate quantitation of an individual peptide, or peptides, from the 1RS1 protein to the amount of another peptide, or peptides, between samples and within samples wherein the amounts as determined by peak area are relative one to another, regardless of the absolute weight to volume or weight to weight amounts of the 1RS1 peptide in the protein preparation from the biological sample, Relative quantitative data about individual signature peak areas between different samples are normalized to the amount of protein analyzed per sample. Relative quantitation can be performed across many peptides from multiple proteins and the IRS I protein simultaneously in a single sample and/or across many samples to gain insight into relative protein amounts, one peptide/protein with respect to other peptides/proteins. 2015215980 24 Aug 2015

Absolute quantitative levels of the IRS1 protein are determined by, for example, the SRM/MRM methodology whereby the SRM/MRM signature peak area of an individual peptide from the IRS I protein in one biological sample is compared to the SRM/MRM signature peak area of a spiked internal standard. In one embodiment, the internal standard is a synthetic version of the same exact IRS I peptide that contains one or more amino acid residues labeled with one or more heavy isotopes. Such isotope labeled internal standards are synthesized so that when analyzed by mass spectrometry it generates a predictable and consistent SRM/MRM signature peak that is different and distinct from the native 1RS1 peptide signature peak and which can be used as a comparator peak. Thus when the internal standard is spiked into a protein preparation from a biological sample in known amounts and analyzed by mass spectrometry, die SRM/MRM signature peak area of the native peptide is compared to the SRM/MRM signature peak area of the internal standard peptide, and this numerical comparison indicates either the absolute molarity and/or absolute weight of the native peptide present in the original protein preparation from the biological sample. Absolute quantitative data for fragment peptides are displayed according to the amount of protein analyzed per sample. Absolute quantitation can be performed across many peptides, and thus proteins, simultaneously in a single sample and/or across many samples to gain insight into absolute protein amounts in individual biological samples and in entire cohorts of individual samples. 3

The SRM/MRM assay method can be used to aid diagnosis of the stage of cancer, for example, directly in patient-derived tissue, such as formalin fixed tissue, and to aid in determining which therapeutic agent would be most advantageous for use in treating that patient. Cancer tissue that is removed from a patient either through surgery, such as for therapeutic removal of partial or entire tumors, or through biopsy procedures conducted to determine the presence or absence of suspected disease, is analyzed to determine whether or not a specific protein, or proteins, and which forms of proteins, are present in that patient tissue. Moreover, the expression level of a protein, or multiple proteins, can be determined and compared to a "normal" or reference level found in healthy tissue. Normal or reference levels of proteins found in healthy tissue may be derived from, for example, the relevant tissues of one or more individuals ihat do not have cancer. Alternatively, normal or reference levels may be obtained for individuals with cancer by analysis of relevant tissues not affected by the cancer. Assays of protein levels (e.g., IRS! levels) can also be used to diagnose the stage of cancer in a patient or subject diagnosed with cancer by employing the IRS1 levels. Levels or amounts of proteins or peptides can be defined as the quantity expressed in moles, mass or weight of a protein or peptide determined by the SRM/MRM assay. The level or amount may be normalized to total the level or amount of protein or another component in the lysate analyzed (e.g., expressed in micromoles/microgram of protein or micrograms /microgram of protein). In addition, the level or amount of a protein or peptide may be determined on volume basis, expressed, for example, in micromolar or nanograms/microliter. The level or amount of protein or peptide as determined by the SRM/MRM assay can also be normalized to the number of cells analyzed. Information regarding IRS1 can thus be used to aid in determining stage or grade of a cancer by correlating the level of the IRS1 protein (or fragment- peptides of the IRS1 protein) with levels observed in normal tissues. Once the stage and/or grade, and/or IRS1 protein expression characteristics of the cancer has been determined, that information can be matched to a list of therapeutic agents (chemical and biological) developed to specifically treat cancer tissue that is characterized by, for example, abnormal expression of the protein or proteinfs) (e.g., IRS1) that were assayed. Matching information from an IRS 1 protein assay to a list of therapeutic agents that specifically targets, for example, the IRS1 protein or cells/tissue expressing the protein, defines what has been termed & personalized medicine approach to treating disease. The assay methods described herein form the foundation of a personalized medicine approach by using analysis of proteins from the patient’s own tissue as a source for diagnostic and treatment decisions. 2015215980 24 Aug 2015 4 2015215980 24 Aug 2015

Detailed Description

In principle, any predicted peptide derived from the IRS! protein, prepared for example by digesting with a protease of known specificity (e.g trypsin), can be used as a surrogate reporter to determine the abundance of IRS I protein in a sample using a mass spectrometry-based SRM/MRM assay. Similarly, any predicted peptide sequence containing an amino acid residue at a site that is known to be potentially modified in the IRS I protein also might potentially be used to assay the extent of modification of the IRS1 protein in a sample. 1RS1 fragment peptides may be generated by a variety of means including by the use of the Liquid Tissue™ protocol provided m US Patent 7,473,532. The Liquid Tissue™ protocol and reagents are capable of producing peptide samples suitable for mass spectroscopic analysis from formalin fixed paraffin embedded tissue by proteolytic digestion of the proteins in the tissue/biological sample. In the Liquid Tissue™ protocol the tissue/biological is heated in a buffer for an extended period of time (e.g., from about 80° C to about 100" C for a period of time from about 10 minutes to about 4 hours) to reverse or release protein cross-linking. The buffer employed is a neutral buffer, (e.g., a Tris-based buffer, or a buffer containing a detergent). Following heat treatment the tissue/biological sample is treated with one or more proteases, including but not limited to trypsin, chymotrypsin, pepsin, and endoproteinase Lys-C for a time sufficient to disrupt the tissue and cellular structure of said biological sample and to liquefy said sample (e.g., a period of time from 30 minutes to 24 hours at a temperature from 37° C to 65° C). The result of the heating and proteolysis is a liquid, soluble, dilutable biomolecute lysate.

Surprisingly, it was found that many potential peptide sequences from the I RSI protein are unsuitable or ineffective for use in mass spectrometry-based SRM/MRM assays for reasons that are not immediately evident. As it was not possible to predict the most suitable peptides for MRM/SRM assay, it was necessary to experimentally identify modified and unmodified peptides in actual Liquid Tissue™ lysates to develop a reliable and accurate SRM/MRM assay for the IRS I protein. While not wishing to be bound by any theory, it is believed that some peptides might, for example, be difficult to detect by mass spectrometry as they do not ionize well or produce fragments distinct from other proteins, peptides may also fail to resolve well in separation (e.g., liquid chromatography), or adhere to glass or plastic ware. 5 2015215980 24 Aug 2015 IRSl peptides found in various embodiments of this disclosure (e.g., Tables I and 2) were derived from the IRS I protein by protease digestion of ail the proteins within a complex Liquid Tissue™ lysate prepared from cells procured from formalin fixed cancer tissue, Unless noted otherwise, in each instance the protease was trypsin. The Liquid Tissue™ lysate was then analyzed by mass spectrometry to determine those peptides derived from the IRSl protein that are detected and analyzed by mass spectrometry. Identification of a specific preferred subset of peptides for mass-spectrometric analysis is based on; I) experimental determination of which peptide or peptides from a protein ionize in mass spectrometry analyses of Liquid Tissue™ lysates, and 2) the ability of the peptide to survive the protocol and experimental conditions used in preparing a Liquid Tissue™ lysate. This latter property extends not only to the amino acid sequence of the peptide but also to the ability of a modified amino acid residue within a peptide to survive in modified form during the sample preparation.

Table 1

Table 1 SEQ ID No. Peptide Sequence SEQ ID NO: 1 EVWQVIIKPKGIGQTK SEQ ID NO: 2 GL6QTKNIH3IYRLCLTSK SEQ ID NO: 3 GSGDYMPMSPKSVSAPQQIINPIR SEQ ID NO: 4 LCGAAGGIENGLNYIDIDLVK SEQ ID NO: 5 INSE AAAWLQLMNIRR SEQ ID NO: 6 LWTNGVGGHHSHVLPHPK SEQ ID NO: 7 NKHIVALYTR SEQ ID NO: 8 PKGLGQTKNLIGIYR SEQ ID NO: 9 RSIPLESCFNINK SEQ ID NO: 10 RTHSAGTSPTITHQK SEQ ID NO: 11 SQSSSNCSNPISVPLRRHHINNPPPSQVGLTR SEQ ID NO: 12 SVSAPQQIINPIRR SEQ ID NO: 13 TISFVKLNSEAAAWIQLMNIR SEQ ID NO: 14 VDTAAQTNSRLAR SEQ ID NO: IS VIRADPQGCRR SEQ ID NO: 16 AASEAGGPARIEYYENEK SEQ ID NO: 17 AAWQEST6VEMGR SEQ ID NO: 18 AAWQESTGVEMGRLGPAPPGAASICR SEQ ID NO: 19 ADPQGCR SEQ ID NO: 20 AMSDEfRPRSK SEQ ID NO: 21 AREQQQQQQPLLHPPEPK SEQ ID NO: 22 ASSDGEGTMSRPASVDGSPVSPSTNR SEQ ID NO: 23 CPSQLQPAPR SEQ ID NO: 24 EEETGTEEYMK 6 2015215980 24 Aug 2015

Table i SEQ ID m. Peptide Sequence SEQ ID NO: 25 CTPGTGLGTSPAIAGDEAASAADLDNR SEQ ID NO: 26 MDIGPGRR SEQ ID NO: 27 FFVLRAASEAGGPAR SEQ ID NO: 28 G6NGHRCTPGTGLGTSPALAGDEAASAADIDNR SEQ ID NO: 29 HHLNNPPPSQVGITR SEQ ID NO: 30 HSAFVPTRSYPEEGLEMHPLER SEQ ID NO: 31 GSGDYMPMSPK SEQ ID NO: 32 VDTAAQTNSR SEQ ID NO: 33 KVGYLRK SEQ ID NO: 34 LARPTRLSLGDPK SEQ ID NO: 35 LHPPLNHSRSIPMPASRCSPSATSPVSLSSSSTSGHGSTSDCLFPR SEQ SO NO: 36 LLYAATADDSSSSTSSDSLGGGYCGAR SEQ ID NO: 37 LSLGDPKASTIPR SEQ ID NO: 38 LSTSSGR SEQ ID NO: 39 PASVD6SPVSPSTNRTHAHR SEQ ID NO: 40 PDSSTLHTDDGYMPMSPGVAPVPSGR SEQ ID NO: 41 PGELGGAPK SEQ ID NO: 42 PRSKSQSSSNCSNPISVPLR SEQ ID NO: 43 PTRLSLGDPKASTLPR SEQ ID NO: 44 QSYVDTSPAAPVSYADMR SEQ ID NO: 45 RHHLNNPPPSQVGLTR SEQ ID NO: 46 HSSETFSSTPSATR SEQ ID NO: 47 RSRTESITATSPASMVGGK SEQ ID NO: 48 RSSEDLSAYASISFQK SEQ ID NO: 49 SIPLESCFNINK SEQ ID NO: 50 SKSQSSSNCSNPISVPLR SEQ ID NO: 51 SRTESITATSPASMVGGK SEQ ID NO: 52 SSASVSGSPSDGGFISSDEYGSSPCDFR SEQ ID NO: 53 SSEDLSAYASJSFQKQPEDR SEQ ID NO: 54 SSFRSVTPDSLGHTPPA SEQ ID NO: 55 GEEELSNYICM6GK SEQ ID NO: 56 SVTPDSLGHTPPAR SEQ ID NO: 57 SYPEEGLEMHPLER SEQ ID NO: 58 TESITATSPASMVGGK SEQ ID NO: 59 VGNTVPFGAGAAVGGGGGSSSSSEDVK SEQ ID NO: 60 VNLSPNRNQSAK SEQ ID NO: 61 GSGDYMPIV1SPK SEQ ID NO: 62 ASSDGEGTMSRPASVDGSPVSPSTNR SEQ ID NO: 63 SVSAPQQIINPSR SEQ ID NO: 64 LClTSKTISFVKLNSEAAAWtQtMNIR SEQ ID NO: 65 LEPSLPHPHHQVLQPHLPR SEQ ID NO: 66 LPGHRHSAFVPTR 7 2015215980 24 Aug 2015

Table .2

Table i SEQID No. Peptide Sequence SEQiD NO: 67 SSEDISAYASISFQK SEQ ID NO: 68 PDSSTlHTDDGYFphosphoyljMPMSPGVAPVPSGR SEQ ID NO: 69 SPGEYtphosphotylJVNIEFGSDQSGYLSGPVAFHSSPSVR SEQID NO: 70 EQQQQQQPLLHPPEPK SEQ ID NO: 71 HSSASFENVWLRPGELGGAPK SEQ ID NO: 72 LEYYENEK SEQ ID NO: 73 INSEAAAWLQLMNIR SEQ ID NO: 74 LSIGDPK SEQ ID NO: 75 NLIGIYR SEQ ID NO: 76 TGIAAEEVSLPR SEQID NO: 77 HLVALYTR

Table 2 SEQ ID NO. Peptide sequence Mono Isotopic Mass Precursor Charge State Precursor mix Transition m/z Ion Type SEQID NO: 22 ASSDGEGTMSRPASVDGSPV SPSTNR 2548.146 2 1275.07996 574.2938 yS 2 857.447 ys 2 1302.628 yl3 2 1373.665 yl4 2 1470.718 yl5 3 850.388977 944.4791 y9 3 1001.5 ylO 3 1116.527 yii 3 1215.596 y!2 3 1302.628 yl3 3 1373.665 yl4 3 1470.718 yl5 SEQID NO:70 EQQQQQQPLLHPPEPK 1923.98 2 962.997009 930.5402 ys 2 1027.593 y9 2 1155.651 ylO 2 1283.71 yll 2 1411.769 yl2 3 642.333984 578.3294 yio 3 704.3721 y6 3 706.388 vl2 3 770.4172 yl3 3 817.4561 V? 3 930.5402 y8 3 1027.593 y9 3 1155.651 ylO SEQ ID NO: 77 HLVALYTR 971.555- 2 486.783997 552.3135 y4 2 623.3506 vs s 2015215980 24 Aug 2015

Table 2 SEQ ID NO. Peptide sequence Mono Isotopic Mass I Precursor Charge State Precursor BS/Z Transition in/z ios 2 722.419 y6 2 835.5031 y? 2 .... 972.562 y8 SEQ ID NO : 71 HSSASFENVWLRPGEIGGAPK 2238.118 2 1120.06504 825.4459 y9 2 1280.71 yl2 2 1379.779 yl3 2 1493.822 yl4 3 747.046021 825.4459 ys 3 981.5471 ylO 3 1008.021 yl9 3 1094,631 yll 3 1280.71 yl2 3 1379.779 yl3 3 1493.822 yl4 SEQ ID NO: 72 LEYYENEK 1086.487 2 $44.25 390.1978 y3 2 682.3037 ys 2 . ......... 845.367 y6 2 974.4096 y7 2 1087.494 y8 SEQ ID NO: 73 LNSEAAAWLQLMNIR 1740.956 2 871.484985 774.4285 y6 2 855.4565 b8 2 887.5126 V7 2 986.581 y8 2 1085.649 y9 2 1156.687 ylO 2 1227.724 yll 2 1298.761 yl2 2 1427.803 yl3 SEQ ID NO: 74 ISLGDPK 728.407 2 355.209992 416.2134 y4 2 529.2975 ys 2 516.3295 y6 2 729.4136 y? 3 354.187012 406.2039 y4 3 507.2516 y5 3 594.2836 ye 3 707.3677 Y7 SEQ ID NO: 75 NLIGIYR 847.492 2 424.752991 451.2658 y3 2 508,2873 y4 | 2 621.3713 y5 2 734.4554 ye 1 2 848,4983 y? SEQ ID NO: 44 QSYVOTSPAAPVSVADMR 1956.883 2 1 979.450989 938.4395 y8 1 9 2015215980 24 Aug 2015

Table 2 SEQ ID NO. Peptide sequence i if 1 s|s Precursor Charge State Precursor in/z Transition tn/z Ion JSffiL 2 1009.477 ys 2 1080.514 yiO 2 1177.567 yll 2 1264.599 yl2 3 6S3302979 655.2863 V5 3 742.3183 y6 3 841.3867 y7 3 938,4395 Y8 3 1009.477 y9 3 1080.514 ylO 3 1177.567 yll SEQ ID NO: S3 SVSAPQQIINPIR 1421.799 2 711.906006 385.2552 y3 2 499.2982 ¥4 2 725.4663 y6 2 853.5248 y7 2 981.5834 y8 2 1078.636 y9 2 1149.673 ylQ 2 1236.705 yll 2 1335.774 yi2 2 1422.806 yl3 SEQ ID NO: 57 SYPEEGLEMHPLER 1685,772 2 843.893005 514.2979 y4 2 718.8453 V12 2 911.4398 V? 2 1024.524 ys 2 1081.545 y9 2 1210.588 YlO 2 1339.63 Yll 2 1436.683 yl2 SEQ ID NO: 76 TGIAAEEVSLPR 1241.662 2 621.838013 272.1712 y2 2 700.3983 y6 2 829.4409 y7 2 900.478 yS 2 971.5151 ¥9 2 1084.599 ylO 2 1141.621 yll 2 1242.668 yl2 were into a sample tube via tissue microdissectbn followed by heating the cells in the Liquid Tissue™ buffer for an extended period of time. Once the formalin-induced cross linking has 10 been negatively affected, the tissue/cells are then digested to completion in a predictable manner using a protease, as for example including but not limited to the protease trypsin. Each protein lysate is turned into a collection of peptides by digestion of intact polypeptides with the protease. Each Liquid Tissue™ lysate was analyzed (e.g., by ion trap mass spectrometry) to perform multiple global proteomic surveys of the peptides where the data was presented as identification of as many peptides as could be identified by mass spectrometry from all cellular proteins present in each protein lysate. An ion trap mass spectrometer or another form of a mass spectrometer that is capable of performing global profiling for identification of as many peptides as possible from a single complex protein/peptide lysate is employed. Ion trap mass spectrometers however may be the best type of mass spectrometer for conducting global profiling of peptides. Although SRM/MRM assay can be developed and performed on any type of mass spectrometer, including a MALDI, ion trap, or triple quadrupole, the most advantageous instrument platform for SRM/MRM assay is often considered to be a triple quadrupole instrument platform. 2015215980 24 Aug 2015

Once as many peptides as possible were identified in a single MS analysis of a single lysate under the conditions employed, then that list of peptides was collated and used to determine the proteins that were detected in that lysate. That process was repeated for multiple Liquid Tissue™ lysates, and the very large list of peptides was collated into a single dataset. That type of dataset can be considered to represent the peptides that can be detected in the type of biological sample that was analyzed (after protease digestion), and specifically in a Liquid Tissue™ lysate of the biological sample, and thus includes the peptides for specific proteins, such as for example the IRS! protein.

In one embodiment, the 1RS1 tryptic peptides identified as useful in the determination of absolute or relative amounts of the IRS1 receptor include one or more, two or mere, three or more, four or more, five or more, six or more, eight or more, or ten or more of the peptides of SEQ ID NO:!, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NQ:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:i 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO.T5, SEQ ID NO: 5 6, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:2I, SEQ ID NQ:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:2S, SEQ ID NO:29, SEQ ID NQ:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:4i, SEQ ID NO;42, SEQ ID NO:43, SEQ ID NQ:44, 11 SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NQ:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:S5, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NG:59, SEQ ID NO:60, SEQ ID NO;61, SEQ ID NO:62, SEQ ID NO:63s SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO;67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:7G, SEQ ID NO:7I, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, each of which are listed in Table I, Each of those peptides was detected by mass spectrometry in Liquid Tissue™ lysates prepared from formalin fixed, paraffin embedded tissue. Thus, each of the peptides in Table 1, or any combination of those peptides (e.g., one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of those peptides recited in Table 1, and particularly combinations with one or more of the peptides found in Table 2) are candidates for use in quantitative SRM/MRM assay for the IRS 1 protein in human biological samples, including directly in formalin fixed patient tissue. 2015215980 24 Aug 2015

The· IRS 1 tryptic peptides listed in Table 1 include those detected from multiple Liquid Tissue™ lysates of multiple different formalin fixed tissues of different human organs including prostate, colon, and breast. Each of those peptides is considered useful for quantitative SRM/MRM assay of the I RSI protein in formalin fixed tissue. Further data analysis of these experiments indicated no preference is observed for any specific peptides from any specific organ site. Thus, each of these peptides is believed to be suitable for conducting SRM/MRM assays of the IRS1 protein on a Liquid Tissue™ lysate from any formalin fixed tissue originating from any biological sample or from any organ site in the body.

In one embodiment the peptides in Table 1, or any combination of those peptides (e.g., one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of those peptides recited in Table 1, and particularly combinations with the peptides also found in Table 2) are assayed by methods that do not rely upon mass spectroscopy, including, but not limited to, immunological methods (e.g.. Western blotting or ELISA). Regardless of how information directed to the amount of the pepiide(s) (absolute or relative) is obtained, the information may be employed in any of the methods described herein, including indicating (diagnosing) the presence of cancer In a subject, determining the stage/grade/slatus of the cancer, providing a prognosis, or determining the therapeutics or treatment regimen for a subject/patient. 12

Embodiments of the present disclosure include compositions comprising one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of the peptides in Table 1, I.n some embodiments, the compositions comprise one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of the peptides in Table 2. Compositions comprising peptides may include one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more peptides that are isotopically labeled. Each of the peptides may be labeled with one or more isotopes selected independently from the group consisting of: i80, i70,34S, ,5N, °C, 2H or combinations thereof. Compositions comprising peptides from the IRS I protein, whether isotope labeled or not, do not need to contain all of the peptides from that protein (e.g., a complete set of tryptic peptides). In some embodiments the compositions do not contain one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more peptides from IRS 1, and particularly peptides appearing in Table ! or Table 2. Compositions comprising peptides may be in the form of dried or lyophilized materials, liquid (e.g., aqueous) solutions or suspensions, arrays, or blots. 2015215980 24 Aug 2015

An important consideration for conducting an SRM/MRM assay is the type of instrument that may be employed in the analysis of the peptides. Although SRM/MRM assays can be developed and performed on any type of mass spectrometer, including a MALDI, ion trap, or triple quadrupole, the most advantageous instrument platform for SRM/MRM assay is often considered to be a triple quadrupole instrument platform. That type of a mass spectrometer may be considered to be the most suitable instrument for analyzing a single isolated target peptide within a very complex protein lysate that may consist of hundreds of thousands to millions of individual peptides from all the proteins contained within a cell.

In order to most efficiently implement SRM/MRM assay for each peptide derived from the IRSS protein it is desirable to utilize information in addition to the peptide sequence in the analysis. That additional information may be used in directing and instructing the mass spectrometer (e.g. a triple quadrupole mass spectrometer), to perform the correct and focused analysis of specific targeted peptide(s), such that the assay may be effectively performed.

The additional information about target peptides in general, and about specific IRS1 peptides, may include one or more of the mono isotopic mass of the peptide, its precursor charge state, the precursor m/z value, the m/z transition ions, and the ion type of each transition ion. Additional peptide information that may be used to develop an SRM/MRM 13 assay for the IRS 1 protein is shown by example for twelve ( !2) of the IRS l peptides from the list in Table 1 and is shown in Table 2. Similar additional information described for these twelve (12) I RSI peptides shown by example in Table 2 may be prepared, obtained, and applied to the analysis of the other peptides contained in Table 1. 2015215980 24 Aug 2015

The method described below was used to: 1) identify candidate peptides from the 1RS1 protein that can be used for a mass spectrometry-based SRM/MRM assay for the IRS1 protein, 2) develop individual SRM/MRM assay, or assays, for target peptides from the IRS! protein in order to correlate and 3) apply quantitative assays to cancer diagnosis and/or choice of optimal therapy.

Assay Method L Identification of SRM/MRM candidate fragment peptides for the IRS1 protein a. Prepare a Liquid Tissue™ protein lysate from a formalin fixed biological sample using a protease or proteases, (that may or may not include trypsin), to digest proteins b. Analyze a!! protein fragments in the Liquid Tissue™ lysate on an ion trap tandem mass spectrometer and identify all fragment peptides from the 1RS1 protein, where individual fragment peptides do not contain any peptide modifications such as phosphorylations or glycosylations c. Analyze all protein fragments in the Liquid Tissue™ lysate on an ion trap tandem mass spectrometer and identify all fragment peptides from the IRS! protein that cany peptide modifications such as for example phosphorylated or glycosylated residues d. All peptides generated by a specific digestion method from the entire, full length IRS! protein potentially can be measured, but preferred peptides used for development of the SRM/MRM assay are those that are identified by mass spectrometry directly in a complex Liquid Tissue™ protein lysate prepared from a formalin fixed biological sample e. Peptides that are specifically modified (phosphoiylated, glycosylated, etc.) in patient tissue and which ionize, and thus detected, in a mass spectrometer when analyzing a Liquid Tissue™ lysate from a formalin fixed biological sample are identified as candidate peptides for assaying peptide modifications of the IRS i protein 2. Mass Spectrometry Assay for Fragment Peptides from 1RS1 Protein 14 a. SRM/MRM assay on a triple quadrupole mass spectrometer for individual fragment peptides identified in a Liquid Tissue™ lysate is applied to peptides from the IRS 1 protein 2015215980 24 Aug 2015 i. Determine optimal retention time for a fragment peptide for optimal chromatography conditions including but not limited to gel electrophoresis, liquid chromatography, capillary electrophoresis, nano reversed phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography 11. Determine, the mono isotopic mass of the peptide, the precursor charge state for each peptide, the precursor m/z value for each peptide, the m/z transition ions for each peptide, and the ion type of each transition Ion for each fragment peptide in order to develop an SRM/MRM assay for each peptide. in. SRM/MRM assay can then be conducted using the information from (i) and (ii) on a triple quadrupole mass spectrometer where each peptide has a characteristic and unique SRM/MRM signature peak that precisely defines the unique SRM/MRM assay as performed on a triple quadrupole mass spectrometer b. Perform SRM/MRM analysis so that the amount of the fragment peptide of the IRSI protein that is detected, as a function of the unique SRM/MRM signature peak area from an SRM/MRM mass spectrometry analysis, can indicate both the relative and absolute amount of the protein in a particular protein lysate. i. Relative quantitation may be achieved by; 1. Determining increased or decreased presence of the IRS 1 protein by comparing the SRM/MRM signature peak area from a given IRS I peptide detected in a Liquid Tissue™ lysate from one formalin fixed biological sample to the same SRM/MRM signature peak area of the same IRSI fragment peptide in at least a second, third, fourth or more Liquid Tissue™ lysates from least a second, third, fourth or more formalin fixed biological samples 2. Determining increased or decreased presence of the IRSI protein by comparing the SRM/MRM signature peak area from a given 15 IRSl peptide detected in a Liquid Tissue™ lysate from one formalin fixed biological sample to SRM/MRM signature peak areas developed from fragment peptides from other proteins, in other samples derived from different and separate biological sources, where the SRM/MRM signature peak area comparison between the 2 samples for a peptide fragment are normalized to amount of protein analyzed in each sample. 2015215980 24 Aug 2015 3. Determining increased or decreased presence of the IRS 1 protein by comparing the SRM/MRM signature peak area for a given IRSl peptide to the SRM/MRM signature peak areas from other fragment peptides derived from different proteins within the same Liquid Tissue™ lysate from the formalin fixed biological sample in order to normalize changing levels of IRSl protein to levels of other proteins that do not change their levels of expression under various cellular conditions. 4. These assays can be applied to both unmodified fragment peptides and for modified fragment peptides of the IRSl protein, where the modifications include but are not limited to phosphorylation and/or glycosylaiion, and where the relative levels of modified peptides are determined in the same manner as determining relative amounts of unmodified peptides, si. Absolute quantitation of a given peptide may be achieved by comparing the SRM/MRM signature peak area for a given fragment peptide from the IRSl protein in an individual biological sample to the SRM/MRM signature peak area of an internal fragment peptide standard spiked into the protein lysate from the biological sample 1. The internal standard is a labeled synthetic version of the fragment peptide from the IRSl protein that is being interrogated. This standard is spiked into a sample in known amounts, and the SRM/MRM signature peak area can be determined for both the internal fragment peptide standard and the native fragment peptide in the biological sample separately, followed by comparison of both peak areas 16 2. This can be applied to unmodified fragment peptides and modified fragment peptides, where the modifications include but are not limited to phosphorylation and/or glyeosylation, and where the absolute levels of modified peptides can be determined in the same manner as determining absolute levels of unmodified peptides. 2015215980 24 Aug 2015 3. Apply Fragment Peptide Quantitation to Cancer Diagnosis and Treatment a. Perform relative and/or absolute quantitation of fragment peptide levels of the I RSI protein and demonstrate that the previously-determined association, as well understood in the field of cancer, of IRS 1 protein expression to the stage/grade/siaius of cancer in patient tumor tissue is confirmed b. Perform relative and/or absolute quantitation of fragment peptide levels of the IRS I protein and demonstrate correlation with clinical outcomes from different treatment strategies, wherein this correlation has already been demonstrated in the field or can be demonstrated in the future through correlation studies across cohorts of patients and tissue from those patients. Once either previously established correlations or correlations derived in the future are confirmed by this assay then the assay method can be used to determine optima! treatment strategy

The information shown in Table 2 is necessary to develop an SRM/MRM assay for quantitation of the IRS I protein on a triplequadrupole mass spectrometer. Specific and unique characteristics about these IRS1 peptides were developed by analysis of all IRS! peptides on both an ion trap and triple quadruple mass spectrometers. That information includes the monoisotopie mass of the peptide, its precursor charge state, the precursor m/z value, the transition m/z values of the precursor, and the ion types of each of the identified transitions. That information must be determined experimentally for each and every candidate SRM/MRM peptide directly in Liquid Tissue™ lysates from formalin fixed tissue; because, interestingly, not all peptides from the IRS1 protein can be detected in such lysates using SRM/MRM as described herein, indicating that IRS l peptides not detected cannot be considered candidate peptides for developing an SRM/MRM assay for use in quantitating pep tides/pro terns directly in Liquid Tissue™ lysates from formalin fixed tissue.

Utilizing this information, quantitative SRM/MRM assays can be developed for the IRS! protein, and assessment of IRS 1 protein levels in tissues based on analysis of formalin fixed patient-derived tissue can provide diagnostic, prognostic, and therapeutically-relevant 17 information about each particular patient, in one embodiment, this disclosure describes a method for measuring the level of the IRS1 protein in a biological sample, comprising detecting and/or quantifying the amount of one or more modified or unmodified IRS 1 fragment peptides in a protein digest prepared from said biological sample using mass spectrometry; and calculating the level of modified or unmodified IRS1 protein in said sample; and wherein said level is a relative level or an absolute level, in a related embodiment, quantifying one or more IRS I fragment peptides comprises determining the amount of the each of the IRS 1 fragment peptides in a biological sample by comparison to an added internal standard peptide of known amount, wherein each of the IRS 1 fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence. In some embodiments the internal standard is an isotopicaiiy labeled internal standard peptide comprises one or more heavy stable isotopes selected from O, '0,' S, N, !3C, 2H or combinations thereof. 2015215980 24 Aug 2015

The method for measuring the level of the IRS1 protein in a biological sample described herein (or fragment peptides as surrogates thereof) may be used as a diagnostic indicator of cancer in a. patient or subject. In one embodiment, the results from measurements of the level of the [RSI protein may be employed to determine the diagnostic stage/grade/status of a cancer by correlating (e.g., comparing) the level of IRS 1 receptor found in a tissue with the level of that protein found in normal and/or cancerous or precancerous tissues.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 18

Claims (11)

1. Claims

   1. A method for measuring the level of the Insulin Receptor Substrate 1 (IRS 1) protein in a human biological sample of formalin fixed tissue, comprising detecting and/or quantifying the amount of a IRS 1 fragment peptide in a protein digest prepared from said human biological sample using mass spectrometry; and calculating the level of IRS 1 protein in said sample; wherein the IRS1 fragment peptide is SEQ ID NO:70 or SEQ ID NO:76, and wherein said level is a relative level or an absolute level.
2. 2. The method of claim 1, further comprising the step of fractionating said protein digest prior to detecting and/or quantifying the amount of said IRS 1 fragment peptide.
3. 3. The method of claim 1 or claim 2, wherein said protein digest comprises a protease digest.
4. 4. The method of any one of claims 1 to 3, wherein the tissue is paraffin embedded tissue.
5. 5. The method of any one of claims 1 to 4, wherein the tissue is obtained from a tumor.
6. 6. The method of any one of claims 1 to 5, further comprising quantifying said IRS1 fragment peptide.
7. 7. The method of claim 6, wherein quantifying said IRS 1 fragment peptide comprises comparing the amount of said IRS 1 fragment peptide in one biological sample to the amount of the same IRS 1 fragment peptide in a different and separate biological sample.
8. 8. The method of claim 7, wherein quantifying said IRS 1 fragment peptide comprises determining the amount of said IRS1 fragment peptide in a biological sample by comparison to an added internal standard peptide of known amount, wherein said IRS 1 fragment peptide in the biological sample is compared to an internal standard peptide having the same amino acid sequence; and wherein the internal standard peptide is an isotopically labeled peptide.
9. 9. The method of any one of claim 1 to 8, wherein detecting and/or quantifying the amount of said IRS 1 fragment peptide in the protein digest indicates the presence of modified or unmodified IRS 1 protein and an association with cancer in the subject.
10. 10. The method of claim 9, further comprising correlating the results of said detecting and/or quantifying the amount of said IRS 1 fragment peptide, or the level of said IRS 1 protein to the diagnostic stage/grade/status of the cancer.
11. 11. The method of claim 10, wherein correlating the results of said detecting and/or quantifying the amount of said IRS 1 fragment peptide, or the level of said IRS 1 protein to the diagnostic stage/grade/status of the cancer is combined with detecting and/or quantifying the amount of other proteins or peptides from other proteins in a multiplex format to provide additional information about the diagnostic stage/grade/status of the cancer.