DETECTION AND QUANTIFICATION OF RAS-RAF-MAPK PATHWAY PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Application

No. 62/465,102, filed February 28, 2017, which is incorporated by reference in its entirety for any purpose.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on February 22, 2018, is named 2018-02-22_01129-0062- 00PCT_Seq_List_ST25 and is 485 kilobytes in size.

FIELD OF THE INVENTION

[0003] This disclosure relates to the field of detection and quantification of RAS-RAF-

MAPK pathway proteins, including adjacent pathway proteins, by immunoprecipitation and mass spectrometry.

BACKGROUND

[0004] The RAS-RAF-MAPK and AKT-mTOR signaling pathways are key regulators of several aspects of normal cell growth and malignant transformation such as cell survival, differentiation, proliferation, metabolism, and motility in response to extracellular cues. These two pathways extensively engage in cross-talk in order to both positively and negatively regulate each other. Because of its importance in cancer, the RAS-MAPK pathway targets have been a focus for drug targeting for last two decades. Rational therapies that target the key regulators of RAS pathway might inhibit tumor growth, survival and spread. See Mendoza MC; Er EE; Blenis I; Trends Biochem Sci. 2011 Jun; 36(6):320-8.

[0005] A major limitation in the detection and quantitation of RAS-RAF-MAPK pathway proteins is the lack of rigorously validated methods and reagents. Currently, only semi-quantitative results from Western blotting, ELISA, and Luminex assays are available. Mass spectrometry (MS) is increasingly becoming the detection methodology of choice for assaying protein abundance and post-translational modifications. However, to date, MS has not been successful in quantifying RAS-RAF-MAPK pathway proteins, possibly due to their low abundance and significant post-translational modification profiles.

[0006] Immunoenrichment or Immunoprecipitation (IP) is commonly used upstream of

MS as an enrichment tool for low-abundant protein targets. See, Gingras et al, Nat. Rev. Mol. Cell. Biol , Aug 2007, 8 (8), 645-54; and Carr, S. A. et al, Mol. Cell. Proteomics Mar 2014, 13 (3), 907-17. The identification of appropriate antibodies for use in IP upstream of MS is important, as not all antibodies that bind to protein will be effective immunoprecipitation tools, and further, not all antibodies that are effective immunoprecipitation tools will lead to successful identification via MS.

SUMMARY

[0007] The present disclosure provides reagents and methods for detecting and quantifying RAS-RAF-MAPK pathway proteins via immunoprecipitation (IP), mass spectrometry (MS), and immunoprecipitation followed by mass spectrometry (IP-MS).

[0008] In some embodiments, methods for immunoprecipitating a RAS-RAF-MAPK pathway protein are provided, comprising contacting a biological sample with any one of the antibodies recited in Table 1. In some embodiments, the antibodies useful in the IP methods comprise the antibodies recited in Table 6. The methods may comprise washing the contacted biological sample to enrich for antibody-protein conjugates. Further methods include detecting the antibody-protein conjugates to determine the RAS-RAF-MAPK pathway protein in the biological sample. In some embodiments, the antibody is labelled. In some embodiments, a detection reagent is provided to the enriched antibody-protein conjugate. In some embodiments the label is biotin and the detection reagent is streptavidin.

[0009] In some embodiments the IP is single-plex. In some embodiments the IP is multi-plex. The antibodies useful in multi-plex IP may comprise the antibodies of Table 6.

[0010] In some embodiments a method for detecting RAS-RAF-MAPK pathway proteins via MS is provided, comprising isolating proteins from a biological sample, digesting the isolated proteins, assaying the digested proteins via mass spectrometry to determine the presence of a peptide for RAS-RAF-MAPK pathway protein(s), and determining the identity of one or more RAS-RAF-MAPK pathway protein(s) in the sample. In some embodiments, the peptide for RAS-RAF-MAPK pathway protein(s) comprises a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. In some embodiments, the peptide is less than or equal to 40 amino acids in length. In some embodiments, the peptide for RAS-RAF-MAPK pathway protein(s) consists of a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. The peptides of SEQ ID NO: 1 - SEQ ID NO: 700 may be labelled. In some embodiments the label on SEQ ID NO: 1 - SEQ ID NO: 700 differs from the label shown on the peptides of SEQ ID NO: 701 - SEQ ID NO: 1400.

[0011] In some embodiments a method for quantifying RAS-RAF-MAPK pathway proteins via MS is provided, comprising isolating proteins from a biological sample, digesting the isolated proteins, assaying the digested proteins via mass spectrometry to determine the presence of a peptide for RAS-RAF-MAPK pathway protein(s), and determining the quantity of one or more RAS-RAF-MAPK pathway protein(s) in the sample. In some embodiments, the peptide for RAS-RAF-MAPK pathway protein(s) comprises a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. In some embodiments, the peptide is less than or equal to 40 amino acids in length. In some embodiments, the peptide for RAS-RAF-MAPK pathway protein(s) consists of a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. The peptides of SEQ ID NO: 1 - SEQ ID NO: 700 may be labelled. In some embodiments the label on SEQ ID NO: 1 - SEQ ID NO: 700 differs from the label shown on the peptides of SEQ ID NO: 701 - SEQ ID NO: 1400. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 13, 15, 18, 28, 31, 47, 48, 51, 127, 128, 132, 133, 141, 142, 143, 144, 171, 173, 177, 180, 181, 182, 183, 190, 192, 213, 219, 220, 224, 226, 231, 233, 234, 235, 236, 258, 270, 295, 297, 300, 301, 302, 304, 306, 308, 310, 347, 348, 353, 362, 392, 393, 405, 416, 429, 443, 446, 451, 467, 483, 487, 501, 502, 506, 507, 510, 512, 521, 522, 523, 528, 572, 580, 588, 591, 598, 603, 615, 617, 635, 636, 655, 657, 660, 662, 670, 678, 683, 691, 692, 696, 698, and 699).

[0012] In some embodiments a method for detecting RAS-RAF-MAPK pathway proteins via IP-MS is provided, comprising treating a biological sample with at least one antibody capable of immunoprecipitating RAS-RAF-MAPK target pathway protein(s) from a biological sample, digesting the isolated proteins, assaying the digested proteins via mass spectrometry to determine the presence of a peptide for RAS-RAF-MAPK pathway protein(s), and determining the identity of one or more RAS-RAF-MAPK pathway protein(s) in the sample. In some embodiments, the peptide for RAS-RAF-MAPK pathway protein(s) comprises a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. In some embodiments the peptide is less than or equal to 40 amino acids in length. In some embodiments, the peptide for RAS- RAF-MAPK pathway protein(s) consists of a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. The peptides of SEQ ID NO: 1 - SEQ ID NO: 700 may be labelled. In some embodiments the label on SEQ ID NO: 1 - SEQ ID NO: 700 differs from the label shown on the peptides of SEQ ID NO: 701 - SEQ ID NO: 1400. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 13, 15, 18, 28, 31, 47, 48, 51, 127, 128, 132, 133, 141, 142, 143, 144, 171, 173, 177, 180, 181, 182, 183, 190, 192, 213, 219, 220, 224, 226, 231, 233, 234, 235, 236, 258, 270, 295, 297, 300, 301, 302, 304, 306, 308, 310, 347, 348, 353, 362, 392, 393, 405, 416, 429, 443, 446, 451, 467, 483, 487, 501, 502, 506, 507, 510, 512, 521, 522, 523, 528, 572, 580, 588, 591, 598, 603, 615, 617, 635, 636, 655, 657, 660, 662, 670, 678, 683, 691, 692, 696, 698, and 699).

[0013] In some embodiments a method for quantifying RAS-RAF-MAPK pathway proteins via IP-MS is provided, comprising treating a biological sample with at least one antibody capable of immunoprecipitating RAS-RAF-MAPK target pathway protein(s) from a biological sample, digesting the isolated proteins, assaying the digested proteins via mass spectrometry to determine the presence of a peptide for RAS-RAF-MAPK pathway protein(s), and determining the quantity of one or more RAS-RAF-MAPK pathway protein(s) in the sample. In some embodiments, the peptide for RAS-RAF-MAPK pathway protein(s) comprises a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. In some embodiments the peptide is less than or equal to 40 amino acids in length. In some embodiments, the peptide for RAS- RAF-MAPK pathway protein(s) consists of a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. The peptides of SEQ ID NO: 1 - SEQ ID NO: 700 may be labelled. In some embodiments the label on SEQ ID NO: 1 - SEQ ID NO: 700 differs from the label shown on the peptides of SEQ ID NO: 701 - SEQ ID NO: 1400. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 13, 15, 18, 28, 31, 47, 48, 51, 127, 128, 132, 133, 141, 142, 143, 144, 171, 173, 177, 180, 181, 182, 183, 190, 192, 213, 219, 220, 224, 226, 231, 233, 234, 235, 236, 258, 270, 295, 297, 300, 301, 302, 304, 306, 308, 310, 347, 348, 353, 362, 392, 393, 405, 416, 429, 443, 446, 451, 467, 483, 487, 501, 502, 506, 507, 510, 512, 521, 522, 523, 528, 572, 580, 588, 591, 598, 603, 615, 617, 635, 636, 655, 657, 660, 662, 670, 678, 683, 691, 692, 696, 698, and 699).

[0014] In some embodiments the RAS-RAF-MAPK pathway target protein is phosphorylated.

[0015] Methods for determining the ratio of phosphorylated to non-phosphorylated

RAS-RAF-MAPK pathway proteins are provided, comprising any of the above IP, MS, or MS- IP methods, wherein a further step of determining the ratio of phosphorylated to non- phosphorylated protein is provided. In some embodiments, the method is an MS-IP method comprising treating a biological sample with one or more antibodies capable of

immunoprecipitating one or more phosphorylated RAS-RAF-MAPK pathway proteins, and separately treating the same biological sample with one or more antibodies capable of immunoprecipitating at least one or more of the same or different non-phosphorylated RAS- RAF-MAPK pathway proteins; digesting the immunoprecipitated RAS-RAF-MAPK pathway proteins; adding a first and a second detectably labelled internal standard peptide of known amount to the digested proteins, wherein the first internal standard peptide has the same amino acid sequence as a phosphorylated RAS-RAF-MAPK pathway peptide used to identify the phosphorylated protein, and the second internal standard peptide has the same amino acid sequence as the non-phosphorylated RAS-RAF-MAPK pathway peptide used to identify the non-phosphorylated protein; assaying the digested protein and internal standards via mass spectrometry to determine the presence and amount of phosphorylated and non-phosphorylated RAS-RAF-MAPK pathway proteins, wherein the RAS-RAF-MAPK pathway peptide comprises a peptide of SEQ ID NO: 1 - SEQ ID NO: 1400, and is less than or equal to 40 amino acids in length; determining the quantity of RAS-RAF-MAPK phosphorylated and non- phosphorylated pathway proteins in the sample, and determining the ratio of phosphorylated to non-phosphorylated pathway proteins. In some embodiments, the peptide for RAS-RAF- MAPK pathway protein(s) consists of a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. The peptides of SEQ ID NO: 1 - SEQ ID NO: 700 may be labelled. In some embodiments the label on SEQ ID NO: 1 - SEQ ID NO: 700 differs from the label shown on the peptides of SEQ ID NO: 701 - SEQ ID NO: 1400. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 13, 15, 18, 28, 31, 47, 48, 51, 127, 128, 132, 133, 141, 142, 143, 144, 171, 173, 177, 180, 181, 182, 183, 190, 192, 213, 219, 220, 224, 226, 231, 233, 234, 235, 236, 258, 270, 295, 297, 300, 301, 302, 304, 306, 308, 310, 347, 348, 353, 362, 392, 393, 405, 416, 429, 443, 446, 451, 467, 483, 487, 501, 502, 506, 507, 510, 512, 521, 522, 523, 528, 572, 580, 588, 591, 598, 603, 615, 617, 635, 636, 655, 657, 660, 662, 670, 678, 683, 691, 692, 696, 698, and 699).

[0016] In some embodiments, the biological sample is human. In some embodiments, the biological sample is non-human. In some embodiments, the biological sample is mammalian. In some embodiments, the biological sample is from rat, mouse, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, cat, or non-human primate.

[0017] In embodiments utilizing a RAS-RAF-MAPK pathway peptide, the peptide may be modified with a detectable label. The detectable label may comprise an isotope, such as a heavy isotope, such as those known to those of skill in the art, including ¹ C, ¹⁵ N, ² H and ¹⁸ 0. In some embodiments, the modified/labelled peptide comprises a peptide of SEQ ID NO: 701- 1400. In some embodiments the peptide is less than or equal to 40 amino acids in length. In some embodiments the modified/labelled peptide consists of a peptide of SEQ ID NO: 701- 1400. In some embodiments the modified/labelled peptide consists of a peptide of SEQ ID NO: 701-1400, wherein the peptide is further modified.

[0018] In some embodiments, the antibody for IP is chosen from the antibodies recited in Table 1. In some embodiments, the antibody for IP is an antibody having the six CDRs of any of the antibodies of Table 1. The antibody may be capable of immunoprecipitating more than one RAS-RAF-MAPK pathway protein. In some embodiments the antibody is labelled or capable of being labelled. The label may be any label known to those of skill in the art including enzymatic and fluorescent labels, such as biotin. In some embodiments more than one antibody is used in a multi-plex IP. In some embodiments, the multi-plex IP comprises the antibodies of Table 6. In some embodiments, the antibodies for multi-plex IP is an antibody having the six CDRs of any of the antibodies in Table 6. [0019] In some embodiments, two or more antibodies are utilized to analyze one biological sample. For example, a first antibody is capable of immunoprecipitating a phosphorylated RAS-RAF-MAPK pathway protein, and a second antibody is capable of immunoprecipitating a nonphosphorylated version of the RAS-RAF-MAPK pathway protein precipitated by the first antibody. In such an instance, a single biological sample may be divided into two aliquots and the first antibody used to immunoprecipitate the first aliquot and the second antibody used to immunoprecipitate the second aliquot. This is appropriate when the second antibody can precipitate both the phospohorylated and non-phosphorylated versions of the RAS-RAF-MAPK protein; it is also appropriate if the second antibody can only precipitate the non-phosphorylated versions. In another instance, the phosphorylated version of the RAS-RAF-MAPK pathway protein may be immunoprecipitated from the sample first (using the first antibody) and evaluated, and then in a later step the non- phosphorylated RAS- RAF-MAPK pathway protein may be immunoprecipitated (using the second antibody) and evaluated. This approach is appropriate if the second antibody can precipitate both the phosphorylated and non-phosphorylated versions of the RAS-RAF-MAPK protein. In some embodiments, a single antibody is capable of immunoprecipitating a phosphorylated and non- phosphorylated RAS-RAF-MAPK pathway protein.

[0020] In some embodiments, the immunoprecipitation comprises treating a sample with a labelled antibody capable of binding to a RAS-RAF-MAPK pathway protein to provide a labelled antibody-protein conjugate. The method may further comprise contacting the labelled antibody-protein conjugate with a capture agent capable of binding to the labelled antibody to isolate the pathway protein from the sample. The label may be biotin and the capture agent may be streptavidin.

[0021] The quantity of a RAS-RAF-MAPK pathway protein may be determined by adding an internal standard peptide of known amount to the digested protein prior to mass spectrometry. In some embodiments, the internal standard peptide has the same amino acid sequence as the RAS-RAF-MAPK pathway peptide. In some embodiments, the internal standard is detectably labeled. The method may further comprises determining the quantity of a RAS-RAF-MAPK pathway peptide by comparison to the internal standard.

[0022] In some embodiments, the internal standard peptide comprises a sequence of

SEQ ID NO: 1 - SEQ ID NO: 1400. In some embodiments the peptide is less than or equal to 40 amino acids in length. In some embodiments, the peptide consists of a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. The peptides of SEQ ID NO: 1 - SEQ ID NO: 700 may be labelled. In some embodiments the label on SEQ ID NO: 1 - SEQ ID NO: 700 differs from the label shown on the peptides of SEQ ID NO: 701 - SEQ ID NO: 1400. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 13, 15, 18, 28, 31, 47, 48, 51, 127, 128, 132, 133, 141, 142, 143, 144, 171, 173, 177, 180, 181, 182, 183, 190, 192, 213, 219, 220, 224, 226, 231, 233, 234, 235, 236, 258, 270, 295, 297, 300, 301, 302, 304, 306, 308, 310, 347, 348, 353, 362, 392, 393, 405, 416, 429, 443, 446, 451, 467, 483, 487, 501, 502, 506, 507, 510, 512, 521, 522, 523, 528, 572, 580, 588, 591, 598, 603, 615, 617, 635, 636, 655, 657, 660, 662, 670, 678, 683, 691, 692, 696, 698, and 699).

[0023] In some embodiments, quantifying the RAS-RAF-MAPK pathway protein comprises comparing an amount of a RAS-RAF-MAPK pathway peptide in the sample to an amount of the same RAS-RAF-MAPK pathway peptide in a control sample.

[0024] Quantifying a RAS-RAF-MAPK pathway protein may comprise comparing an amount of a RAS-RAF-MAPK pathway peptide to an internal standard peptide of known amount, wherein both the peptide in the biological sample and the internal standard peptide comprise SEQ ID NO: 1-SEQ ID NO: 1400, wherein the standard peptide is detectably labeled, and wherein the peptide is less than or equal to 40 amino acids long. In some embodiments, the standard peptide consists of a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. The peptides of SEQ ID NO: 1 - SEQ ID NO: 700 may be labelled. In some embodiments the label on SEQ ID NO: 1 - SEQ ID NO: 700 differs from the label shown on the peptides of SEQ ID NO: 701 - SEQ ID NO: 1400. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 13, 15, 18, 28, 31, 47, 48, 51, 127, 128, 132, 133, 141, 142, 143, 144, 171, 173, 177, 180, 181, 182, 183, 190, 192, 213, 219, 220, 224, 226, 231, 233, 234, 235, 236, 258, 270, 295, 297, 300, 301, 302, 304, 306, 308, 310, 347, 348, 353, 362, 392, 393, 405, 416, 429, 443, 446, 451, 467, 483, 487, 501, 502, 506, 507, 510, 512, 521, 522, 523, 528, 572, 580, 588, 591, 598, 603, 615, 617, 635, 636, 655, 657, 660, 662, 670, 678, 683, 691, 692, 696, 698, and 699).

[0025] In some embodiments the mass spectrometry is selected from tandem mass spectrometry and discovery mass spectrometry. The targeted mass spectrometry may be selected from multiple reaction monitoring (MRM), selected reaction monitoring (SRM), and parallel reaction monitoring (PRM), or combinations thereof.

[0026] In some embodiments the biological sample is selected from isolated human cells, plasma, serum, whole blood, CSF, urine, sputum, tissue, and tumorous tissue.

[0027] In some embodiments, the method further comprises quantifying the relative amount of RAS-RAF-MAPK pathway protein. In some embodiments, the method further comprises quantifying the absolute amount of RAS-RAF-MAPK pathway protein.

[0028] In some embodiments, the digesting comprises a protease or chemical digest. In some embodiments the digestion may be single or sequential. The protease digestion may comprise trypsin, chymotrypsin, AspN, GluC, LysC, LysN, ArgC, proteinase K, pepsin, clostripain, elastase, GluC biocarb, LysC/P, LysN promise, protein endopeptidase, staph protease or thermolysin.

[0029] The chemical cleavage may comprise CNBr, iodosobenzoate or formic acid.

[0030] In some embodiments the digestion is a protease digest with trypsin.

[0031] In some embodiments the methods further comprise desalting after digestion and prior to mass spectrometry.

[0032] The RAS-RAF-MAPK pathway protein may be chosen from proteins listed in

Table 4

[0033] In some embodiments, the RAS-RAF-MAPK pathway protein is

phosphorylated.

[0034] In some embodiments, the concentration of RAS-RAF-MAPK protein that may be detected ranges from about 0.04 fmol to about 2000 fmol.

[0035] In some embodiments, the lower limit of detection is no more than about 0.04,

0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 1.23 fmol. The lower limit of detection may be from of about 0.04 - 1.23 fmol.

[0036] In some embodiments the lower limit of quantification is no more than about

0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, or 11.11 fmol. The lower limit of quantification may be from about 0.04 - 11.11 fmol.

[0037] Kits comprising one or more antibodies capable of immunoprecipitating a RAS-

RAF-MAPK pathway protein are encompassed.

[0038] Kits comprising one or more antibodies capable of immunoprecipitating a RAS-

RAF-MAPK pathway protein, and reagents useful for performing mass spectrometry to detect a RAS-RAF-MAPK pathway protein are also provided.

[0039] Also encompassed are kits comprising one or more antibodies capable of immunoprecipitating a RAS-RAF-MAPK pathway target protein, and reagents useful for performing mass spectrometry to quantify a RAS-RAF-MAPK pathway protein.

[0040] The antibody to be included in the kit may be selected from any one or more of the antibodies recited in Table 1. In some embodiments the antibody is labelled or capable of being labelled. The label may be any label known to those of skill in the art including enzymatic and fluorescent labels, such as biotin. In some embodiments, the kit comprises more than one antibody. In some embodiments, the kit comprises two or more of the antibodies selected from the antibodies recited in Table 6. [0041] The kits may further comprise a RAS-RAF-MAPK pathway peptide. In some embodiments, the peptide comprises a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. In some embodiments the peptide is less than or equal to 40 amino acids in length. In some embodiments, the peptide consists of a sequence of SEQ ID NO: 1 - SEQ ID NO: 1400. The peptides of SEQ ID NO: 1 - SEQ ID NO: 700 may be labelled. In some embodiments the label on SEQ ID NO: 1 - SEQ ID NO: 700 differs from the label shown on the peptides of SEQ ID NO: 701 - SEQ ID NO: 1400. In some embodiments, the peptide comprises or consists of a peptide selected from the peptides shown in Table 5 (SEQ ID Nos: 13, 15, 18, 28, 31, 47, 48, 51, 127, 128, 132, 133, 141, 142, 143, 144, 171, 173, 177, 180, 181, 182, 183, 190, 192, 213, 219, 220, 224, 226, 231, 233, 234, 235, 236, 258, 270, 295, 297, 300, 301, 302, 304, 306, 308, 310, 347, 348, 353, 362, 392, 393, 405, 416, 429, 443, 446, 451, 467, 483, 487, 501, 502, 506, 507, 510, 512, 521, 522, 523, 528, 572, 580, 588, 591, 598, 603, 615, 617, 635, 636, 655, 657, 660, 662, 670, 678, 683, 691, 692, 696, 698, and 699).

[0042] In some embodiments, the kit may comprise at least one peptide selected from peptides of SEQ ID NO: 701 - SEQ ID NO: 1400, wherein the peptide is less than or equal to 40 amino acids. In one embodiment, the kit comprises at least one peptide consisting of SEQ ID NO: 701 - SEQ ID NO: 1400.

[0043] The peptides provided in the kit may be detectably labeled or capable of being modified to be detectably labeled. In some embodiments, the kit may comprise at least one peptide selected from peptides of SEQ ID NO: 1 - SEQ ID NO: 700, wherein the peptide is detectably labeled or capable of being modified to be detectably labeled.

[0044] In some embodiments, the kit further comprises a protease or chemical agent capable of digesting an immunoprecipitated protein sample. The protease agent may be trypsin, chyniotrypsin, AspN, GluC, LysC, LysN, ArgC, proteinase K, pepsin, clostripain, elastase, GluC biocarb, LysC/P, LysN promise, protein endopeptidase, Staph protease or thermolysin. The chemical agent may be CNBr, iodosobenzoate or formic acid.

[0045] In some embodiments, the kit may comprise some or all of the following items: single or multiple biotinylated antibody mix, streptavidin magnetic beads (such as Pierce Strepavidin Magnetic Beads), IP-MS cell lysis buffer, IP-MS wash buffer(s), IP-MS elution buffer, positive control lysates, low binding tubes, reduction/alkylation reagents for MS sample prep, digestion buffer for MS sample prep, trypsin, buffer to stop trypsin digestion (such as 10% trifiuoroacetic acid), AQUA (Absolute QUAntification) heavy peptide mix, diluent for peptides (such as 0.1% trifiuoroacetic acid/5% acetonitrile), matrix (light crude peptides spiked into 6 proteins digest mix), and memory device (such as a USB stick) including the instrument method, skyline software template, and instructions. [0046] The kits may be utilized to detect RAS-RAF-MAPK pathway proteins listed in

Table 4

[0047] The RAS-RAF-MAPK protein to be detected and quantified by the kits may be phosphorylated.

BRIEF DESCRIPTION OF THE FIGURES

[0048] FIGURE 1 shows one representative workflow for an immunoprecipitation- enriched mass spectrometry assay to identify RAS-RAF-MAPK pathway proteins.

[0049] FIGURE 2 shows results from an experiment to enrich for low abundant RAS-

RAF-MAPK pathway proteins from HCT116 or BT549 cells.

[0050] FIGURE 3 shows quantitation limits of peptides for RAS-RAF-MAPK pathway proteins.

[0051] FIGURE 4 shows the results of a multiplex immunoprecipitation plus nanoLC-

MS/MS assay for 12 RAS-RAF-MAPK pathway proteins panel 1.

[0052] FIGURE 5 shows the results of a multiplex immunoprecipitation plus nanoLC-

MS/MS assay for 12 RAS-RAF-MAPK pathway proteins panel 2.

[0053] FIGURE 6 shows the results of a multiplex immunoprecipitation plus nanoLC-

MS/MS assay for 13 RAS-RAF-MAPK pathway proteins Panel 3.

[0054] FIGURE 7 shows a summary of RAS-RAF-MAPK pathway proteins identified and quantified in HCT116 cell lines, with and without immunoprecipitation enrichment, using the mass spec methods.

DETAILED DESCRIPTION

[0055] This description and exemplary embodiments should not be taken as limiting.

For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about," to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. [0056] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0057] As used herein, an "RAS-RAF-MAPK pathway protein" includes, but is not limited to, BRAF (UniProtKB - PI 5056), HRAS (UniProtKB - P01112), KRAS (UniProtKB P01116), NRAS (UniProtKB - P01111), MAP2K1 (UniProtKB - Q02750), MAP2K2 (UniProtKB - P36507), MAPK1 (UniProtKB - P28482), MAPK3 (UniProtKB - P27361), MAPK8

(UniProtKB - P45983), MAPK9 (UniProtKB - P45984), PIK3CA (UniProtKB - P42336), RPS6KA1 (also known as p90S6K) (UniProtKB - Q15418), and TP53 (UniProtKB - P04637).

[0058] As used herein "protein", "peptide", and "polypeptide" are used

interchangeably throughout to mean a chain of amino acids wherein each amino acid is connected to the next by a peptide bond. In some embodiments, when a chain of amino acids consists of about two to forty amino acids, the term "peptide" is used. However, the term "peptide" should not be considered limiting unless expressly indicated.

[0059] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments so long as they exhibit the desired immunoprecipitating activity. As such, the term antibody includes, but is not limited to, fragments that are capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', di-scFv, sdAb (single domain antibody) and (Fab')2 (including a chemically linked F(ab')2). Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment. Pepsin treatment yields a F(ab')2 fragment that has two antigen-binding sites. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, goat, horse, sheep, chicken, etc. Furthermore, for all antibody constructs provided herein, variants having the sequences from other organisms are also contemplated, such as CDR-grafted antibodies or chimeric antibodies. Antibody fragments also include either orientation of single chain scFvs, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies, etc. Antibody fragments also include nanobodies (sdAb, an antibody having a single, monomelic domain, such as a pair of variable domains of heavy chains, without a light chain). An antibody fragment can be referred to as being a specific species in some embodiments (for example, human scFv or a mouse scFv). This denotes the sequences of at least part of the non-CDR regions, rather than the source of the construct. The antibodies are referred to by reference to name and catalog reference. The skilled artisan, holding this name and catalog information, is capable of determining the sequence of the antibody, and therefore this application encompasses any antibody having at least partial sequence of a reference antibody so long as the antibody maintains its ability to immunoprecipitate its antigen protein. In some embodiments, the antibodies comprise antibodies having the same CDRs as the antibodies provided in Table 1 or Table 6.

[0060] "Immunoprecipitate" or "immunoprecipitation" refers to any antibody-driven enrichment step, whether or not an actual precipitate is formed. Thus, immunoprecipitation includes any immunoenrichment step.

[0061] Mass spectrometry (MS) is a primary technique for analysis of proteins on the basis of their mass-to-charge ratio (m/z). MS techniques generally include ionization of compounds and optional fragmentation of the resulting ions, as well as detection and analysis of the m/z of the ions and/or fragment ions followed by calculation of corresponding ionic masses. A "mass spectrometer" generally includes an ionizer and an ion detector. "Mass spectrometry," "mass spec," "mass spectroscopy," and "MS" are used interchangeably throughout.

[0062] "Targeted mass spectrometry," also referred to herein as "targeted mass spec,"

"targeted MS," and "tMS" increases the speed, sensitivity, and quantitative precision of mass spec analysis. Non-targeted mass spectrometry, sometimes referred to as "data-dependent scanning," "discovery MS," and "dMS" and targeted mass spec are alike in that in each, analytes (proteins, small molecules, or peptides) are infused or eluted from a reversed phase column attached to a liquid chromatography instrument and converted to gas phase ions by electrospray ionization. Analytes are fragmented in the mass spec (a process known as tandem MS or MS/MS), and fragment and parent masses are used to establish the identity of the analyte. Discovery MS analyzes the entire content of the MS/MS fragmentation spectrum. In contrast, in targeted mass spectrometry, a reference spectrum is used to guide analysis to only a few selected fragment ions rather than the entire content.

[0063] "Multiple reaction monitoring," "MRM," "selected reaction monitoring," and

"SRM" are used interchangeably throughout to refer to a type of targeted mass spectrometry that relies on a unique scanning mode accessible on triple-quadrupole (QQQ) instruments. See, e.g., Chambers et al, Expert Rev. Proteomics, 1-12 (2014).

[0064] "Parallel Reaction Monitoring," and "PRM" are used interchangeably herein to describe another type of targeted mass spec wherein the second mass analyzer used in SRM (quadrupole) is substituted by a high resolution orbitrap mass analyzer in PRM. Unlike SRM, which allows the measuring of one single transition at a given point in time, PRM allows parallel monitoring in one MS/MS spectrum. PRM also allows for the separation of ions with close m/z values (i.e., within a 10 ppm range), and may therefore allow for lower limits of detection and quantification (LOD or LLOD and LOQ or LLOQ).

[0065] The methods disclosed herein may be applied to any type of MS analysis. The disclosure is not limited by the specific equipment or analysis used. The use of any equipment with the intent of analyzing the m/z of a sample would be included in the definition of mass spectrometry. Non-limiting examples of MS analysis and/or equipment that may be used include electrospray ionization, ion mobility, time-of-flight, tandem, ion trap, MRM, SRM, MRM/SRM, PRM, and Orbitrap. The disclosure is neither limited by the type of ionizer or detector used in the MS analysis nor by the specific configuration of the MS. The disclosure is not limited to use with specific equipment or software. The disclosure is not limited to the equipment and software described in the Examples.

[0066] In some embodiments, methods of immunoprecipitating a RAS-RAF-MAPK pathway protein are provided, comprising contacting a biological sample with at least one antibody recited in Table 1. The immunoprecipitating method may be single-plex or multiplex. A "single-plex" IP utilizes one antibody per assay, whereas a "multi-plex" IP utilizes more than one antibody per assay.

[0067] In some embodiments, an IP-MS method for detecting and quantifying phosphorylated and non-phosphorylated RAS-RAF-MAPK pathway proteins is provided. The methods may comprise contacting a biological sample with at least one antibody recited in Table 1, digesting the immunoprecipitated protein(s), and assaying the digested proteins via mass spectrometry. The IP and MS may be single-plex or multi-plex. A "single-plex" MS refers to monitoring a single peptide in a single MS run, whereas a "multi-plex" MS refers to monitoring more than one target peptides in a single MS run.

[0068] Table 1 provides a listing of antibodies useful in the IP and IP-MS methods.

Table 2 provides a listing of antibodies that are known to bind to their antigen RAS-RAF- MAPK protein, but were found to be less useful in the IP and IP-MS methods.

Table 1: List of IP to MS validated antibodies for RAS-RAF-MAPK Pathway Proteins

ALK Antibody Thermo Fisher Scientific/35-4300

ALK Antibody Thermo Fisher Scientific/51-3900

ARAF Antibody Thermo Fisher Scientific/PA 1-21440

ARAF Antibody Thermo Fisher Scientific/PA5-17657

BRAF Antibody Cell Signaling Technology/14814S

BRAF Antibody Thermo Fisher Scientific/PA5- 14926

BRAF Antibody Thermo Fisher Scientific/PA5- 14929

BRAF Antibody Thermo Fisher Scientific/MA5-15000

CALM1 Antibody Millipore/05-173

CAMK2A Antibody Thermo Fisher Scientific/PA5- 17095

CCND1 Antibody Thermo Fisher Scientific/MAl-34820

CCND1 Antibody Thermo Fisher Scientific/PA 1-37530

CCND1 Antibody Thermo Fisher Scientific/MA5-14512

Cyclin Dl Antibody Cell Signaling Technology /2922S

Cyclin Dl Antibody Millipore/ABE52

Cyclin Dl Antibody Thermo Fisher Scientific/MAl-12296

Cyclin Dl Antibody Thermo Fisher Scientific/PAl-29132

Cyclin Dl Antibody Thermo Fisher Scientific/MAl-26755

Cyclin Dl Antibody Thermo Fisher Scientific/AHF0082

Cyclin Dl Antibody Thermo Fisher Scientific/33-3500

Cyclin Dl Antibody Thermo Fisher Scientific/PA5-32373

Cyclin Dl / Bel- 1 Antibody Thermo Fisher Scientific/PA5-16232

CDH1 Antibody Cell Signaling Technology/14472S

CDH1 Antibody Thermo Fisher Scientific/13-5700

CDH1 Antibody Thermo Fisher Scientific/13-1700

CDH1 Antibody Thermo Fisher Scientific/33-4000

CDH1 Antibody Thermo Fisher Scientific/PA5-32282

CDH1 Antibody Thermo Fisher Scientific/PA5-32178

CDH1 Antibody Thermo Fisher Scientific/MAl-06300

CDH1 Antibody Thermo Fisher Scientific/MA5-14458

CDH1 Antibody Thermo Fisher Scientific/MA5- 12547

CDH2 Antibody Thermo Fisher Scientific/PAl-20359

CDH2 Antibody Thermo Fisher Scientific/MAl-159

CDH2 Antibody Thermo Fisher Scientific/MA5-16324

CDK2 Antibody Thermo Fisher Scientific/MA5-13725

CDK2 Antibody Thermo Fisher Scientific/AHZ0142

CDKNlA/p21 Antibody Cell Signaling Technology /883 IS

CDKNlA/p21 Antibody Millipore/05-655

CDKNlA/p21 Antibody Millipore/05-345

CDKNlA/p21 Antibody Thermo Fisher Scientific/PA5 -25960

CDKNlA/p21 Antibody Thermo Fisher Scientific/PA5- 12644

CDKNlA/p21 Antibody Thermo Fisher Scientific/MA5- 14949

CDKNlA/p21 Antibody Thermo Fisher Scientific/MAl-4800

CDKNlA/p21 Antibody Thermo Fisher Scientific/MAl- 12303

CDKNlA/p21 Antibody Thermo Fisher Scientific/MAl -19271

CDKNlA/p21 Antibody Thermo Fisher Scientific/PA 1-30399 CDKNlA/p21 Antibody Thermo Fisher Scientific/MA 1-91243

CDKNlA/p21 Antibody Thermo Fisher Scientific/MAl-33926

CDKNlA/p21 Antibody Thermo Fisher Scientific/AHZ0422

CDKNlA/p21 Antibody Thermo Fisher Scientific/710182

CDKNlA/p21 Antibody Thermo Fisher Scientific/701151

CDKN1B Antibody Cell Signaling Technology /3688S

CDKN1B Antibody Thermo Fisher Scientific/PA5-18480

CDKN1B Antibody Thermo Fisher Scientific/PA5- 17946

CDKN1B Antibody Thermo Fisher Scientific/PA5-27188

CDKN1B Antibody Thermo Fisher Scientific/PA5-16717

CDKN1B Antibody Thermo Fisher Scientific/PA5-32530

CDKN1B Antibody Thermo Fisher Scientific/PAl-38351

CDKN2A/p 14ARF Antibody Abcam/ab 11048

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-31409

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/MAl-20219

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/MA5- 14260

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-46021

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-16662

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-16639

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PA5-32529

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-38334

CDKN2A/pl6INK4a Antibody Abcam/abl6123

CDKN2A/pl6INK4a Antibody Abcam/abl25282

CDKN2A/pl6INK4a Antibody Thermo Fisher Scientific/PA 1-9025

CDKN2A/pl6INK4a Antibody Thermo Fisher Scientific/PA5-20379

CTNNA1 Antibody Thermo Fisher Scientific/MAl-2000

CTNNAl Antibody Thermo Fisher Scientific/ 13 -9700

CTNNBl Antibody Cell Signaling Technology /9582S

CTNNBl Antibody Cell Signaling Technology/8814S

CTNNBl Antibody Thermo Fisher Scientific/PA5- 19469

CTNNBl Antibody Thermo Fisher Scientific/MAl-10056

CTNNBl Antibody Thermo Fisher Scientific/MAl-34999

CTNNBl Antibody Thermo Fisher Scientific/PA5- 16762

CTNNBl Antibody Thermo Fisher Scientific/PA5- 16429

CTNNBl Antibody Thermo Fisher Scientific/MA 1-301

CTNNBl Antibody Thermo Fisher Scientific/MAl-300

CTNNBl Antibody Thermo Fisher Scientific/MA 1-2001

CTNNBl Antibody Thermo Fisher Scientific/13-8400

CTNNBl Antibody Thermo Fisher Scientific/71-2700

CTNNBl Antibody Thermo Fisher Scientific/44206M

CTNNBl Antibody Thermo Fisher Scientific/PAl-37255

CTNNBl Antibody Thermo Fisher Scientific/MA5- 14461

DDR2 Antibody Cell Signaling Technology/12133S

DUSP22 Antibody Thermo Fisher Scientific/PA5-33488

DUSP4 Antibody Cell Signaling Technology/5149

DUSP4 Antibody Thermo Fisher Scientific/PA5- 15549 DUSP6 Antibody Thermo Fisher Scientific/PA5-21570

DUSP6 Antibody Thermo Fisher Scientific/PA5-17107

EGFR Antibody Thermo Fisher Scientific/MA5-13697

EGFR Antibody Thermo Fisher Scientific/MA5-13319

EGFR Antibody Thermo Fisher Scientific/MA5- 13269

EGFR Antibody Thermo Fisher Scientific/MA5-13070

EGFR Antibody Thermo Fisher Scientific/MA5-13048

EGFR Antibody Thermo Fisher Scientific/MA5- 12880

EGFR Antibody Thermo Fisher Scientific/MA5-12875

EGFR Antibody Thermo Fisher Scientific/PAl-1110

EGFR Antibody Thermo Fisher Scientific/700308

EGFR Antibody Thermo Fisher Scientific/700994

EGFR Antibody Thermo Fisher Scientific/MA5-13877

EGFR Antibody Thermo Fisher Scientific/MA5- 13343

EGFR Antibody Thermo Fisher Scientific/MA5- 14485

EIF2A Antibody Proteintech/11233-1-AP

EIF2A Antibody Abcam/ab 102786

EIF2A Antibody Thermo Fisher Scientific/PA5-34795

EIF4E Antibody Cell Signaling Technology /9742S

EIF4E Antibody Thermo Fisher Scientific/PA5-11797

EIF4E Antibody Thermo Fisher Scientific/MAl-089

EIF4EBP1 Antibody Cell Signaling Technology /9644S

EIF4EBP1 Antibody Cell Signaling Technology /4923S

EIF4EBP1 Antibody Thermo Fisher Scientific/PA5-11830

EIF4EBP1 Antibody Thermo Fisher Scientific/PA5 -28947

EIF4EBP1 Antibody Thermo Fisher Scientific/PA5-20135

ERBB3 Antibody Cell Signaling Technology/12708S

ERBB3 Antibody Thermo Fisher Scientific/PA 1-30497

ERBB3 Antibody Thermo Fisher Scientific/MA5-13053

ERBB3 Antibody Thermo Fisher Scientific/MA5- 13040

ERBB3 Antibody Thermo Fisher Scientific/MA5- 12867

ERBB3 Antibody Thermo Fisher Scientific/MA5- 12675

ERBB3 Antibody Thermo Fisher Scientific/MA5-16349

ERBB3 Antibody Thermo Fisher Scientific/PAl -86644

ERBB3 Antibody Thermo Fisher Scientific/PA 1-9040

ERBB3 Antibody Thermo Fisher Scientific/PA5-32346

ERBB4 Antibody Cell Signaling Technology /4795S

ERBB4 Antibody Thermo Fisher Scientific/PAl -20922

ERBB4 Antibody Thermo Fisher Scientific/MA5- 12888

ERBB4 Antibody Thermo Fisher Scientific/PA5- 16642

ERBB4 Antibody Thermo Fisher Scientific/PA5-32347

ESR1 Antibody Cell Signaling Technology /8644S

ESR1 Antibody Millipore/06-935

ESR1 Antibody Thermo Fisher Scientific/MAl-12692

ESR1 Antibody Thermo Fisher Scientific/MAl-23214

ESR1 Antibody Thermo Fisher Scientific/MA 1-25623 ESR1 Antibody Thermo Fisher Scientific/PAl-309

ESR1 Antibody Thermo Fisher Scientific/MA5-13065

ESR1 Antibody Thermo Fisher Scientific/PA5- 16440

ESR1 Antibody Thermo Fisher Scientific/MA5-13191

ESR1 Antibody Thermo Fisher Scientific/MA5- 13304

ESR1 Antibody Thermo Fisher Scientific/PA5-32397

FBX07 Antibody Novus Bio/NBPl-55051

FBX07 Antibody Millipore/ABN1038

FBX07 Antibody Millipore/MABN447

FBX07 Antibody Thermo Fisher Scientific/PA5-42300

FBX07 Antibody Thermo Fisher Scientific/PA5-27589

FGFR1 Antibody Cell Signaling Technology /9740S

FGFR1 Antibody Thermo Fisher Scientific/PA5-14650

FGFR1 Antibody Thermo Fisher Scientific/PA5- 14648

FGFR3 Antibody Thermo Fisher Scientific/MA5- 14843 c-Fos Antibody Thermo Fisher Scientific/PA5- 17489 c-fos Antibody Thermo Fisher Scientific/MA5-15055 c-Fos Antibody Thermo Fisher Scientific/MAl-21190 c-Fos Antibody Thermo Fisher Scientific/38-4950 c-Fos Antibody Thermo Fisher Scientific/PAl-37437

FOXOl Antibody Thermo Fisher Scientific/82358

FOXOl Antibody Thermo Fisher Scientific/MA5-14846

FOXOl Antibody Thermo Fisher Scientific/PA5-18696

FOXOl Antibody Thermo Fisher Scientific/PA5-32412

FOX03 Antibody Cell Signaling Technology/12829S

FOX03 Antibody Thermo Fisher Scientific/PA5-19519

FOX03 Antibody Thermo Fisher Scientific/PA 1-9042

FOX03 Antibody Thermo Fisher Scientific/PA 1-31322

FOX03 Antibody Thermo Fisher Scientific/PAl-805

FOX03 Antibody Thermo Fisher Scientific/PA5-23191

FOX03 Antibody Thermo Fisher Scientific/PA5-32413

HER-2 / ErbB2 Antibody Thermo Fisher Scientific/PAl-12361

HER-2 / ErbB2 Antibody Thermo Fisher Scientific/PA 1-37426

HER-2 / ErbB2 Antibody Antibody Thermo Fisher Scientific/PA5-16305

HER-2 / ErbB2 Antibody (3B5) Antibody Thermo Fisher Scientific/MA5-13675

HER-2 / ErbB2 Antibody (9G6.10) Antibody Thermo Fisher Scientific/MA5- 12759

HER-2 / ErbB2 Antibody (e2-4001 + 3B5) Thermo Fisher Scientific/MA5-14057

Antibody

HER-2 / ErbB2 Antibody (K.929.9) Antibody Thermo Fisher Scientific/MA5-15050

HER-2 / ErbB2 Antibody (L26) Antibody Thermo Fisher Scientific/MA5-13679

HER-2/ERBB-2 Antibody Thermo Fisher Scientific/MAl-12691

HER-2/ERBB-2 Antibody Thermo Fisher Scientific/AHOlOl l

IQGAP1 Antibody Abcam/ab33542

IQGAP1 Antibody Thermo Fisher Scientific/33-8900

JAK1 Antibody Cell Signaling Technology /3332S

JAK1 Antibody Cell Signaling Technology /3344S JAK2 Antibody Cell Signaling Technology /323 OS

JAK2 Antibody Thermo Fisher Scientific/ AH01352

JAK2 Antibody Thermo Fisher Scientific/44-406G

MAP2K1 Antibody Thermo Fisher Scientific/MA5-15093

MAP2K1 Antibody Thermo Fisher Scientific/PAl -29035

MAP2K2 Antibody Abcam/ab28834

MAP2K2 Antibody Thermo Fisher Scientific/PA5- 17678

MAP2K2 Antibody Thermo Fisher Scientific/PA5-27635

MAPK1 Antibody Thermo Fisher Scientific/PAl -37640

MAPK1 Antibody Thermo Fisher Scientific/PA5-32396

MAPK1 Antibody Abcam/ab7948

MAPK1 Antibody Thermo Fisher Scientific/PAl -30388

MAPK1 Antibody Thermo Fisher Scientific/PAl -21016

MAPK1/3 Antibody Proteintech/66192-l-Ig

MAPK11 Antibody Abcam/ab80116

MAPK8 Antibody Thermo Fisher Scientific/LF-PA0047

MAPK8 Antibody Thermo Fisher Scientific/35-9800

MAPK9 Antibody Thermo Fisher Scientific/PA5 -28664

MAPK9 Antibody Thermo Fisher Scientific/PA5- 17634

MAPK9 Antibody Thermo Fisher Scientific/MA 1-21411

MET Antibody Cell Signaling Technology /8198S

MET Antibody Thermo Fisher Scientific/PA5-17148

MET Antibody Thermo Fisher Scientific/MA5- 14847

MET Antibody Thermo Fisher Scientific/PA5-27235

MET Antibody Thermo Fisher Scientific/PAl -29042

MET Antibody Thermo Fisher Scientific/PAl -28026

MET Antibody Thermo Fisher Scientific/71-8000

MET Antibody Thermo Fisher Scientific/37-0100

MET Antibody Thermo Fisher Scientific/18-7366

MET Antibody Thermo Fisher Scientific/700261

MET Antibody Thermo Fisher Scientific/PA5-32357

MTOR Antibody Thermo Fisher Scientific/ PA1-188

MTOR Antibody Thermo Fisher Scientific/ PA1-518

NF1 Antibody Abcam/ab 17963

NF1 Antibody Millipore/05-1430

NF1 Antibody Millipore/07-730

NF1 Antibody Thermo Fisher Scientific/PAl -46246

NOTCH 1 Antibody Cell Signaling Technology /3608S

NOTCH 1 Antibody Thermo Fisher Scientific/41-3500

NOTCH 1 Antibody Thermo Fisher Scientific/PA5-32522

PAK1 Antibody Thermo Fisher Scientific/PA5- 17798

PAK1 Antibody Thermo Fisher Scientific/71-9300

PAK1,2,3 Antibody Thermo Fisher Scientific/PA5- 17789 pan MAPK8/9 Antibody Proteintech/51153-l-AP

pan MAPK8/9 Antibody Thermo Fisher Scientific/AH01362

Pan-Cadherin Antibody Thermo Fisher Scientific/PA5-17526 Pan-Cadherin Antibody Thermo Fisher Scientific/MA5-15036

Pan-Cadherin Antibody Thermo Fisher Scientific/PA5- 16766

Pan-Cadherin Antibody Thermo Fisher Scientific/PA5- 16481

Pan-Cadherin Antibody Thermo Fisher Scientific/71-7100

Pan-Cadherin Antibody Thermo Fisher Scientific/PAl-37199

PARP1 Antibody Thermo Fisher Scientific/MA5-15031

PARP1 Antibody Thermo Fisher Scientific/PA5-16561

PGR Antibody Cell Signaling Technology /8757S

PGR Antibody Thermo Fisher Scientific/MAl-12626

PGR Antibody Thermo Fisher Scientific/MA5-12658

PGR Antibody Thermo Fisher Scientific/MA5- 12642

PGR Antibody Thermo Fisher Scientific/PA5- 16709

PGR Antibody Thermo Fisher Scientific/MAl-411

PGR Antibody Thermo Fisher Scientific/MA5-14842

PGR Antibody Thermo Fisher Scientific/PA 1-38491

PGR Antibody Thermo Fisher Scientific/PA5-17180

PGR Antibody Thermo Fisher Scientific/MA5- 14841

PI3KCA Antibody Millipore/09-481

PI3KCA Antibody Millipore/07-658

PI3KCA Antibody Thermo Fisher Scientific/MA5- 14870

PIK3R2 Antibody Thermo Fisher Scientific/MAl-90048

PSMG1 Antibody Cell Signaling Technology/13378S

PSMG1 Antibody Abcam/ab83527

PSMG1 Antibody Thermo Fisher Scientific/PA5- 19076

RAF1 Antibody Millipore/07-396

RAF1 Antibody Thermo Fisher Scientific/PA5 -28652

RAF1 Antibody Thermo Fisher Scientific/MA5- 17162

Ras- pan Antibody Thermo Fisher Scientific/MAl-012

RASA1 Antibody Abcam/ab2922

RASA1 Antibody Millipore/06-157

RASA1 Antibody Thermo Fisher Scientific/MA4-001

RASGRF1 Antibody Proteintech/12958-l-AP

RASGRF2 Antibody Origene/TA590561

RASGRF2 Antibody Abcam/abl21577

Ras-N Antibody Cell Signaling Technology /3965S

Ras-N Antibody Thermo Fisher Scientific/PA5- 18888

Ras-N Antibody Thermo Fisher Scientific/PA5-34560

Ras-v-H Antibody Thermo Fisher Scientific/33-7200

RICTOR Antibody Cell Signaling Technology /9476S

RICTOR Antibody Abcam/ab70373

RICTOR Antibody Thermo Fisher Scientific/PA5-14107

RICTOR Antibody Thermo Fisher Scientific/PA5- 17451

RICTOR Antibody Thermo Fisher Scientific/MA5-15006

RICTOR Antibody Thermo Fisher Scientific/PA5-18370

RICTOR Antibody Thermo Fisher Scientific/PA5-21582

RICTOR Antibody Thermo Fisher Scientific/PAl-41209 RPS6KB2 Antibody Cell Signaling Technology/14130S

RPS6KB2 Antibody Abcam/abl31526

RPS6KB2 Antibody Abcam/ab76960

RPS6KB2 Antibody Abcam/ab37699

RPS6KB2 Antibody Thermo Fisher Scientific/PA5- 15234

Raptor Antibody Millipore/09-217

Raptor Antibody Cell Signaling Technology /2280S

Raptor Antibody Millipore/05-1470

Raptor Antibody Thermo Fisher Scientific/42-4000

Raptor Antibody Thermo Fisher Scientific/PA5-20125

RSK1 (RPS6KA1) Antibody Cell Signaling Technology /8408S

RSK1 (RPS6KA1) Antibody Thermo Fisher Scientific/PA5-23665

RSK1 (RPS6KA1) Antibody Thermo Fisher Scientific/PA5-17168

RSK1 (RPS6KA1) Antibody Thermo Fisher Scientific/PA5-29215

RSK1 (RPS6KA1) Antibody Thermo Fisher Scientific/PA5-21699

RSK2 (RPS6KA3) Antibody Millipore/06-918

RSK2 (RPS6KA3) Antibody Thermo Fisher Scientific/PA5- 17169

RSK2 (RPS6KA3) Antibody Thermo Fisher Scientific/PA5-29214

RSK2 (RPS6KA3) Antibody Thermo Fisher Scientific/38-6800

RSK2 (RPS6KA3) Antibody Thermo Fisher Scientific/PAl-4694

RSK2 + RSK3 Antibody Thermo Fisher Scientific/MA5-17212

SMAD3 Antibody Thermo Fisher Scientific/51-1500

SMAD4 Antibody R&D Systems/AF2097-SP

SMAD4 Antibody Thermo Fisher Scientific/PA5- 14845

SMAD4 Antibody Thermo Fisher Scientific/PA5-16191

SMAD4 Antibody Thermo Fisher Scientific/PA5-34806

SMAD4 Antibody Thermo Fisher Scientific/PAl-31458

SMAD4 Antibody Thermo Fisher Scientific/MA5- 14300

SMAD4 Antibody Thermo Fisher Scientific/701682

SOS1 Antibody Cell Signaling Technology/12409

SOS1 Antibody Cell Signaling Technology /5890S

SOS1 Antibody Millipore/07-337

SPRED1 Antibody Millipore/ABS186

SPRED1 Antibody Genetex/GTX85376

SPRED1 Antibody Thermo Fisher Scientific/PA5-20618

SPRED1 Antibody Thermo Fisher Scientific/PA5-20617

SPRED2 Antibody Millipore/ABS198

SPRY1 Antibody Cell Signaling Technology/13013S

SPRY1 Antibody Thermo Fisher Scientific/PA5- 18289

SPRY1 Antibody Thermo Fisher Scientific/40- 1800

SPRY2 Antibody Millipore/07-524

SPRY2 Antibody Cell Signaling Technology/14954S

SPRY3 Antibody LifeSpan BioSciences/LS-C335646

SPRY4 Antibody Abcam/ab82298

SPRY4 Antibody Thermo Fisher Scientific/PA5 -26959

SPRY4 Antibody Thermo Fisher Scientific/PA5-14349 STAT1 Antibody Thermo Fisher Scientific/AHO0832

STAT3 Antibody Cell Signaling Technology /9139S

STAT3 Antibody Thermo Fisher Scientific/PA5-18562

STAT3 Antibody Thermo Fisher Scientific/PAl-9105

STAT3 Antibody Thermo Fisher Scientific/MAl-13042

STAT3 Antibody Thermo Fisher Scientific/13-7000

STK11 Antibody Thermo Fisher Scientific/MA5- 14942

TP53 Antibody Thermo Fisher Scientific/MAl-12648

TP53 Antibody Thermo Fisher Scientific/MAl-12549

TP53 Antibody Thermo Fisher Scientific/MAl-19055

TP53 Antibody Thermo Fisher Scientific/MA5- 15244

TP53 Antibody Thermo Fisher Scientific/MA5-14067

TP53 Antibody Thermo Fisher Scientific/MA5-12453

TP53 Antibody Thermo Fisher Scientific/MA5-11296

TP53 Antibody Thermo Fisher Scientific/AHO0152

TP53 Antibody Thermo Fisher Scientific/MA5-12557

TP53 Antibody Thermo Fisher Scientific/ 13 -4100

TP53 Antibody Thermo Fisher Scientific/13-4000

TP53 Antibody Thermo Fisher Scientific/49-1031

TP53 Antibody Thermo Fisher Scientific/MA5-14467

TP53 Antibody Thermo Fisher Scientific/MA5-14516

Phospho-TP53 (pSerl5) Antibody Thermo Fisher Scientific/700439

Phospho-TP53 (pSerl5) Antibody Thermo Fisher Scientific/MA5- 15229

Phospho-MAPKl/3 (pThr202/pTyr204) Thermo Fisher Scientific/MA5-15173

Antibody

Phospho-MAPKl/3 (pThr202/pTyr204) Thermo Fisher Scientific/PAl -26678

Antibody

Phospho-MAPKl/3 (pThr202/pTyr204) Cell Signaling Technology /9101

Antibody

Phospho-RAFl (pSer259) Antibody Thermo Fisher Scientific/44502

Phospho-RAFl (pSer259) Antibody Cell Signaling Technology /9421

Phospho-RPS6KA3 (pSer380) Antibody Thermo Fisher Scientific/44928G

Phospho-RPS6KA3 (pSer380) Antibody Cell Signaling Technology/11989

Phospho-MAPK8/9 pThrl83/pTyrl85 Antibody Cell Signaling Technology /9251

Phospho-MAPK8/9 pThrl83/pTyrl85 Antibody Thermo Fisher Scientific/MA5- 14943

Phospho-c-Raf (Ser338) Antibody Thermo Fisher Scientific/MA5-15176

Phospho-MEKl (Ser298) Antibody Cell Signaling Technology/98195

Phospho-MEKl/MEK2 (Ser217, Ser221) Thermo Fisher Scientific/MA5-15016

Antibody

Phospho-ATF2 (Thr71) Antibody Thermo Fisher Scientific/MA5-15088

Phospho-ATF-2 (Thr71) Antibody Cell Signaling Technology /9221

Phospho-SAPK/JNK (Thrl83/Tyrl85) Cell Signaling Technology /4668

Antibody

Table 2: List of IP to MS less successful antibodies for RAS-RAF-MAPK Pathway Proteins Antibody Name Company /Catalog Number

ALK Antibody Thermo Fisher Scientific/PA5- 14564

ARAF Antibody Thermo Fisher Scientific/PA5- 14924

ARAF Antibody Thermo Fisher Scientific/PA5-21354

BRAF Antibody Thermo Fisher Scientific/PA5- 19690

BRAF Antibody Thermo Fisher Scientific/PA5- 14933

BRAF Antibody Thermo Fisher Scientific/PA5- 14928

BRAF Antibody Thermo Fisher Scientific/MA5-15495

BRAF Antibody Thermo Fisher Scientific/MA5-15317

BRAF Antibody Millipore/07-453

CALM1 Antibody Thermo Fisher Scientific/PA5-11662

CALM1 Antibody Thermo Fisher Scientific/MA3-917

CALM1 Antibody Abcam/ab2860

CAMK2A Antibody Thermo Fisher Scientific/PA5-14315

CAMK2A Antibody Thermo Fisher Scientific/PA5- 14033

CAMK2A Antibody Thermo Fisher Scientific/A 16650

CAMK2A Antibody Thermo Fisher Scientific/A14012

CAMK2A Antibody Thermo Fisher Scientific/PA5-38239

CAMK2A Antibody Thermo Fisher Scientific/PA5-19128

CAMK2A Antibody Thermo Fisher Scientific/13-7300

CAMK2A Antibody Abcam/ab22609

CAMK2A Antibody Abcam/ab50202

CAMK2A Antibody Proteintech/20666-l-AP

CCND1 Antibody Thermo Fisher Scientific/PA5-24173

CCND1 Antibody Thermo Fisher Scientific/PA5-12255

Cyclin Dl Antibody Thermo Fisher Scientific/MAl-10324

Cyclin Dl / Bel- 1 Antibody Thermo Fisher Scientific/PA5-16777

Cyclin Dl / Bcl-1 Antibody Thermo Fisher Scientific/PA5-16607

Cyclin Dl / Bcl-1 Antibody Thermo Fisher Scientific/MA5- 12707

Cyclin Dl / Bcl-1 Antibody Thermo Fisher Scientific/710428

Cyclin Dl / Bcl-1 Antibody Thermo Fisher Scientific/701421

CDH1 Antibody Thermo Fisher Scientific/MAl-10192

CDH1 Antibody Thermo Fisher Scientific/MA5- 14408

CDH1 Antibody Thermo Fisher Scientific/MA5-11496

CDH1 Antibody Thermo Fisher Scientific/710161

CDH1 Antibody Thermo Fisher Scientific/701134

CDH1 Antibody Thermo Fisher Scientific/PA5-11548

CDH1 Antibody Thermo Fisher Scientific/PA5-11549

CDH1 Antibody Thermo Fisher Scientific/MA5-17013

CDH1 Antibody Thermo Fisher Scientific/MAl-06302

CDH1 Antibody Thermo Fisher Scientific/MAl-06304

CDH1 Antibody Thermo Fisher Scientific/MA 1-37202

CDH1 Antibody Thermo Fisher Scientific/PA5-32339

CDH1 Antibody Thermo Fisher Scientific/MAl-34228

CDH2 Antibody Thermo Fisher Scientific/PA5- 19486

CDH2 Antibody Thermo Fisher Scientific/PA5-29570 CDH2 Antibody Thermo Fisher Scientific/MAl-2002

CDH2 Antibody Thermo Fisher Scientific/33-3900

CDH2 Antibody Thermo Fisher Scientific/PA5-32283

CDKNlA/p21 Antibody Thermo Fisher Scientific/PA5-34805

CDKNlA/p21 Antibody Thermo Fisher Scientific/PA5-14456

CDKNlA/p21 Antibody Thermo Fisher Scientific/PA5- 12647

CDKNlA/p21 Antibody Thermo Fisher Scientific/PA5- 12646

CDKNlA/p21 Antibody Thermo Fisher Scientific/PAl-4185

CDKNlA/p21 Antibody Thermo Fisher Scientific/MAl-91045

CDKNlA/p21 Antibody Thermo Fisher Scientific/33-7000

CDKN1B Antibody Thermo Fisher Scientific/MAl -12302

CDKN1B Antibody Thermo Fisher Scientific/AHZ0452

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-16661

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-31212

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-30670

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-29914

CDKN2A/p 14ARF Antibody Thermo Fisher Scientific/PAl-28218

CDKN2A/pl6INK4a Antibody Thermo Fisher Scientific/MA5- 17093

CTNNAl Antibody Thermo Fisher Scientific/71-1200

CTNNBl Antibody Thermo Fisher Scientific/44207M

CTNNBl Antibody Thermo Fisher Scientific/MA5-15569

DDR2 Antibody Thermo Fisher Scientific/PA5- 14751

DDR2 Antibody Thermo Fisher Scientific/PA5 -27752

DDR2 Antibody Thermo Fisher Scientific/PA5-27730

DDR2 Antibody Thermo Fisher Scientific/PA 1-31993

DUSP1 Antibody Thermo Fisher Scientific/PA5- 15593

DUSP1 Antibody Thermo Fisher Scientific/PA5- 17973

DUSP22 Antibody Thermo Fisher Scientific/PA5 -29427

DUSP22 Antibody Thermo Fisher Scientific/PA5-33489

DUSP22 Antibody Thermo Fisher Scientific/PA5-33487

DUSP22 Antibody Proteintech/51005-2- AP

DUSP22 Antibody Abnova/H00056940-D01

DUSP4 Antibody Thermo Fisher Scientific/MA5-17225

DUSP4 Antibody Abcam/ab72593

DUSP4 Antibody Abcam/ab60229

DUSP6 Antibody Thermo Fisher Scientific/PA5-22162

DUSP6 Antibody Thermo Fisher Scientific/PA5-15552

DUSP6 Antibody Thermo Fisher Scientific/PAl-28321

DUSP6 Antibody Proteintech/10433-l-AP

EIF2A Antibody Thermo Fisher Scientific/PA5-41916

EIF2A Antibody Thermo Fisher Scientific/PA5-29593

EIF2A Antibody Thermo Fisher Scientific/PA5-27366

EIF2A Antibody Thermo Fisher Scientific/MAl-079

EIF2A Antibody Thermo Fisher Scientific/AH01182

EIF2A Antibody Thermo Fisher Scientific/AHO0802

EIF4E Antibody Thermo Fisher Scientific/PA5-24582 EIF4E Antibody Thermo Fisher Scientific/PA5-41751

EIF4EBP1 Antibody Thermo Fisher Scientific/PA5-30679

EIF4EBP1 Antibody Thermo Fisher Scientific/AH01382

EIF4EBP1 Antibody Thermo Fisher Scientific/MA5-15314

ERBB3 Antibody Thermo Fisher Scientific/MA5-13008

ERBB3 Antibody Thermo Fisher Scientific/MAl-860

ERBB3 Antibody Thermo Fisher Scientific/PA5-14636

ERBB3 Antibody Thermo Fisher Scientific/PAl-31989

ERBB3 Antibody Thermo Fisher Scientific/MAl-22819

ERBB4 Antibody Thermo Fisher Scientific/PA5 -29272

ERBB4 Antibody Thermo Fisher Scientific/PA5-14638

ERBB4 Antibody Thermo Fisher Scientific/PA5-14637

ERBB4 Antibody Thermo Fisher Scientific/PAl -20923

ERBB4 Antibody Thermo Fisher Scientific/PA5- 16789

ERBB4 Antibody Thermo Fisher Scientific/MA5-13789

ERBB4 Antibody Thermo Fisher Scientific/MA5-13016

ERBB4 Antibody Thermo Fisher Scientific/MAl-861

ERBB4 Antibody Thermo Fisher Scientific/PA5-32758

ERBB4 Antibody Millipore/05-478

ESR1 Antibody Thermo Fisher Scientific/PAl -308

ESR1 Antibody Thermo Fisher Scientific/MAl-12600

ESR1 Antibody Thermo Fisher Scientific/MA5- 12328

ESR1 Antibody Thermo Fisher Scientific/417000

ESR1 Antibody Thermo Fisher Scientific/MA5-15268

ESR1 Antibody Thermo Fisher Scientific/PA5-34577

ESR1 Antibody Thermo Fisher Scientific/PAl -37651

ESR1 Antibody Thermo Fisher Scientific/49-1002

ESR1 Antibody Thermo Fisher Scientific/MAl-310

FGFR1 Antibody Thermo Fisher Scientific/PA5-28564

FGFR1 Antibody Thermo Fisher Scientific/PA5 -25979

FGFR1 Antibody Thermo Fisher Scientific/PA5-24590

FGFR1 Antibody Thermo Fisher Scientific/PA5- 14649

FGFR1 Antibody Thermo Fisher Scientific/PA5- 14646

FGFR1 Antibody Thermo Fisher Scientific/PA5-18344

FGFR1 Antibody Thermo Fisher Scientific/PA5-27139

FGFR1 Antibody Thermo Fisher Scientific/PAl -24762

FGFR1 Antibody Thermo Fisher Scientific/MAl-90880

FGFR1 Antibody Thermo Fisher Scientific/MA 1-26256

FGFR1 Antibody Thermo Fisher Scientific/ 13 -3100

FGFR1 Antibody Thermo Fisher Scientific/PA5-32403

FGFR1 Antibody Thermo Fisher Scientific/PAl-37682

FGFR1 Antibody Millipore/05-1134

FGFR1 Antibody Millipore/05-149

FGFR2 Antibody Thermo Fisher Scientific/PA5- 14654

FGFR2 Antibody Thermo Fisher Scientific/PA5- 14653

FGFR2 Antibody Thermo Fisher Scientific/PA5- 14652 FGFR2 Antibody Thermo Fisher Scientific/PA5- 14651

FGFR2 Antibody Thermo Fisher Scientific/PA5-18914

FGFR2 Antibody Thermo Fisher Scientific/PA5 -29426

FGFR2 Antibody Thermo Fisher Scientific/PAl-31990

FGFR2 Antibody Thermo Fisher Scientific/P A 1-24764

FGFR2 Antibody Thermo Fisher Scientific/P A 1-24763

FGFR2 Antibody Thermo Fisher Scientific/P A5-32404

FGFR2 Antibody Thermo Fisher Scientific/PAl-37685

FGFR3 Antibody Thermo Fisher Scientific/P A5- 19782

FGFR3 Antibody Thermo Fisher Scientific/P A5-14655

FGFR3 Antibody Thermo Fisher Scientific/PA5-34574

FGFR3 Antibody Thermo Fisher Scientific/P A5-32405

FGFR3 Antibody Thermo Fisher Scientific/PAl-37687 c-fos Antibody Thermo Fisher Scientific/P A5- 13531 c-Fos Antibody Thermo Fisher Scientific/PA 1-24330 c-Fos Antibody Thermo Fisher Scientific/PA 1-830 c-Fos Antibody Thermo Fisher Scientific/OSCOOOl lG c-Fos Antibody Thermo Fisher Scientific/MA5- 17076 c-Fos Antibody Genetex/GTX27963

FOXOl Antibody Thermo Fisher Scientific/P A5-23132

FOXOl Antibody Thermo Fisher Scientific/MAl-23236

FOXOl Antibody Thermo Fisher Scientific/MAl-23234

FOXOl Antibody Thermo Fisher Scientific/MAl-23232

FOXOl Antibody Thermo Fisher Scientific/MA 1-23230

FOXOl Antibody Thermo Fisher Scientific/PAl-804

FOXOl Antibody Thermo Fisher Scientific/MA5- 17078

FOXOl Antibody Thermo Fisher Scientific/P A5-20972

FOXOl/3 Antibody Thermo Fisher Scientific/P A5-38276

FOXO 1/3/4 Antibody Thermo Fisher Scientific/P A5-36084

FOXO 1/3/4 Antibody Thermo Fisher Scientific/P A5-38274

FOX03 Antibody Thermo Fisher Scientific/PA5-27145

FOX03 Antibody Thermo Fisher Scientific/P A5-20973

HER-2 / ErbB2 Antibody Thermo Fisher Scientific/PA5-14635

HER-2 / ErbB2 Antibody Thermo Fisher Scientific/P A5- 14634

HER-2 / ErbB2 Antibody Thermo Fisher Scientific/P A5- 14632

HER-2 / ErbB2 Antibody Thermo Fisher Scientific/MA5-16724

HER-2 / ErbB2 Antibody Antibody Thermo Fisher Scientific/P A5- 16774

HER-2 / ErbB2 Antibody Antibody Thermo Fisher Scientific/P A5 -20740

HER-2 / ErbB2 Antibody (40H87L57), Thermo Fisher Scientific/700635 ABfinity™ Rabbit Monoclonal Antibody

HER-2 / ErbB2 Antibody (7H3L20), Thermo Fisher Scientific/701399 ABfinity™ Rabbit Monoclonal Antibody

HER-2 / ErbB2 Antibody (7HCLC), ABfinity™ Thermo Fisher Scientific/710452 Rabbit Oligoclonal Antibody

HER-2 / ErbB2 Antibody (ICR55) Antibody Thermo Fisher Scientific/MAl-82367

HER-2 / ErbB2 Antibody (L87 + 2ERB19) Thermo Fisher Scientific/MA5-11976 Antibody HER-2 / ErbB2 Antibody (N12) Antibody Thermo Fisher Scientific/MA5- 12998

HER-2 / ErbB2 Antibody (N28) Antibody Thermo Fisher Scientific/MA5-13032

IQGAP1 Antibody Thermo Fisher Scientific/PA5-19189

IQGAP1 Antibody Thermo Fisher Scientific/PA5-34368

IQGAP1 Antibody Cell Signaling Technology /2293S

JAK1 Antibody Thermo Fisher Scientific/PA5-34811

JAK1 Antibody Thermo Fisher Scientific/PA5-27583

JAK2 Antibody Thermo Fisher Scientific/PA5-11267

JAK2 Antibody Thermo Fisher Scientific/PA5-34649

KSR1 Antibody Thermo Fisher Scientific/PA5-14937

KSR1 Antibody Thermo Fisher Scientific/PA5- 14027

KSR1 Antibody Thermo Fisher Scientific/MAl-25285

KSR1 Antibody Abcam/abl4516

MAP2K1 Antibody Thermo Fisher Scientific/PA5 -27693

MAP2K1 Antibody Thermo Fisher Scientific/MAl-095

MAP2K1 Antibody Thermo Fisher Scientific/PA5-15161

MAP2K1 Antibody Thermo Fisher Scientific/PA5- 17677

MAP2K1 Antibody Thermo Fisher Scientific/PA 1-21730

MAP2K1 Antibody Thermo Fisher Scientific/PA5-16556

MAP2K1 Antibody Thermo Fisher Scientific/ 13 -3500

MAP2K1 Antibody Thermo Fisher Scientific/710446

MAP2K2 Antibody Thermo Fisher Scientific/PA5 -29291

MAP2K2 Antibody Thermo Fisher Scientific/PA5 -27640

MAP2K2 Antibody Thermo Fisher Scientific/700829

MAP2K2 Antibody Thermo Fisher Scientific/PA5-15167

MAP2K2 Antibody Thermo Fisher Scientific/PA 1 -29852

MAP2K2 Antibody Thermo Fisher Scientific/PAl-41411

MAP2K2 Antibody Abcam/ab71828

MAP2K2 Antibody Cell Signaling Technology/9147S

MAPK1 Antibody Thermo Fisher Scientific/PA5- 14088

MAPK1 Antibody Thermo Fisher Scientific/PA5-14414

MAPK1 Antibody Thermo Fisher Scientific/PA 1-29167

MAPK1 Antibody Thermo Fisher Scientific/PA5-18329

MAPK1 Antibody Thermo Fisher Scientific/PA5-17710

MAPK1 Antibody Thermo Fisher Scientific/PAl-28196

MAPK1 Antibody Thermo Fisher Scientific/PA5-29636

MAPK1 Antibody Thermo Fisher Scientific/MAl-099

MAPK1 Antibody Thermo Fisher Scientific/LF-MA0193

MAPK1 Antibody Thermo Fisher Scientific/MA5- 15304

MAPK1/3 Antibody Thermo Fisher Scientific/MA5-15134

MAPK1/3 Antibody Thermo Fisher Scientific/82380

MAPK1/3 Antibody Thermo Fisher Scientific/61-7400

MAPK1/3 Antibody Thermo Fisher Scientific/44-654G

MAPK1/3 Antibody Thermo Fisher Scientific/13-6200

MAPK1/3 Antibody Thermo Fisher Scientific/710318

MAPK1/3 Antibody Thermo Fisher Scientific/710214 MAPK1/3 pan Antibody Thermo Fisher Scientific/PAl-25987

MAPK1/3 pan Antibody Thermo Fisher Scientific/MA5- 15227

MAPK11 Antibody Thermo Fisher Scientific/PA5-29302

MAPK11 Antibody Thermo Fisher Scientific/PA5- 14425

MAPK11 Antibody Thermo Fisher Scientific/PA5- 14424

MAPK11 Antibody Thermo Fisher Scientific/PA5-14050

MAPK11 Antibody Thermo Fisher Scientific/PAl -29850

MAPK11 Antibody Thermo Fisher Scientific/33-8700

MAPK11 Antibody Thermo Fisher Scientific/PAl -41154

MAPK3 Antibody Thermo Fisher Scientific/PAl -30387

MAPK3 Antibody Thermo Fisher Scientific/MAl-13041

MAPK3 Antibody Thermo Fisher Scientific/13-8600

MAPK3 Antibody Thermo Fisher Scientific/701183

MAPK3 Antibody Thermo Fisher Scientific/PA5-14413

MAPK3 Antibody Thermo Fisher Scientific/PAl-21015

MAPK3 Antibody Thermo Fisher Scientific/PA5-32911

MAPK3 Antibody Thermo Fisher Scientific/PA5-32912

MAPK3 Antibody Thermo Fisher Scientific/PA5-32913

MAPK3 Antibody Thermo Fisher Scientific/PAl -41413

MAPK3 Antibody Thermo Fisher Scientific/PAl -9041

MAPK3 Antibody Thermo Fisher Scientific/PAl -37638

MAPK3 Antibody Thermo Fisher Scientific/PA5-32395

MAPK8 Antibody Thermo Fisher Scientific/PA5-21343

MAPK8 Antibody Thermo Fisher Scientific/PA5-25151

MAPK8 Antibody Thermo Fisher Scientific/PA5-14418

MAPK8 Antibody Thermo Fisher Scientific/PA5- 17889

MAPK8 Antibody Thermo Fisher Scientific/44-690G

MAPK8 Antibody Thermo Fisher Scientific/710075

MAPK8 Antibody Thermo Fisher Scientific/MA5-17117

MAPK9 Antibody Thermo Fisher Scientific/PA5-28262

MAPK9 Antibody Thermo Fisher Scientific/MA5-15183

MAPK9 Antibody Thermo Fisher Scientific/AH01162

MAPK9 Antibody Thermo Fisher Scientific/PAl -41234

MET Antibody Thermo Fisher Scientific/MA5-15198

MET Antibody Thermo Fisher Scientific/MA5-23760

NF1 Antibody Thermo Fisher Scientific/MAl-085

NF1 Antibody Thermo Fisher Scientific/MAl-084

NF1 Antibody LifeSpan BioSciences/LS-C2721

NOTCH 1 Antibody Thermo Fisher Scientific/MAl-91405

NOTCH 1 Antibody Thermo Fisher Scientific/MA5-11961

NOTCH 1 Antibody Thermo Fisher Scientific/PA5-23181

NOTCH 1 Antibody Thermo Fisher Scientific/MAl-81888

PAK1 Antibody Thermo Fisher Scientific/PA5-29256

PAK1 Antibody Thermo Fisher Scientific/PA5 -29224

PAK1 Antibody Thermo Fisher Scientific/PA5-34696

PAK1 Antibody Thermo Fisher Scientific/PA5-15115 PAK1 Antibody Thermo Fisher Scientific/PA5-18557

PAK1 Antibody Thermo Fisher Scientific/PAl-28385

PAK1,2 Antibody Thermo Fisher Scientific/PA5-36918

PAK1,2 Antibody Thermo Fisher Scientific/PA5-38693

PAK1,2,3 Antibody Thermo Fisher Scientific/PA5-36102

PAK1,2,3 Antibody Thermo Fisher Scientific/PA5-38311

PAK1,2,3 Antibody Thermo Fisher Scientific/PA5-38310 pan MAP2K Antibody Thermo Fisher Scientific/PAl- 10034 pan MAP2K Antibody Thermo Fisher Scientific/PA5-31917 pan MAP2K Antibody Thermo Fisher Scientific/MA5-15162 pan MAP2K Antibody Thermo Fisher Scientific/MA5-15135 pan MAP2K Antibody Thermo Fisher Scientific/PA5-31918 pan MAPK8/9 Antibody BD Biosciences/554285

Pan-Cadherin Antibody Thermo Fisher Scientific/PA5- 19479

PARP1 Antibody Thermo Fisher Scientific/PA5-34803

PARP1 Antibody Thermo Fisher Scientific/PA5-34802

PARP1 Antibody Thermo Fisher Scientific/PA5-13386

PARP1 Antibody Thermo Fisher Scientific/PA5-27219

PARP1 Antibody Thermo Fisher Scientific/PA5- 16452

PARP1 Antibody Thermo Fisher Scientific/A21969

PARP1 Antibody Thermo Fisher Scientific/436400

PARP1 Antibody Thermo Fisher Scientific/MAl-41020

PARP1 Antibody Thermo Fisher Scientific/PA5-32561

PARP1 Antibody Thermo Fisher Scientific/PAl -38414

PGR Antibody Thermo Fisher Scientific/MA5- 12653

PGR Antibody Thermo Fisher Scientific/MAl-412

PGR Antibody Thermo Fisher Scientific/PA5-16435

PGR Antibody Thermo Fisher Scientific/PA5-31314

PGR Antibody Thermo Fisher Scientific/MAl-23267

PGR Antibody Thermo Fisher Scientific/MAl-23268

PGR Antibody Thermo Fisher Scientific/PA5-34849

PGR Antibody Thermo Fisher Scientific/PA5- 17226

PI3KCA Antibody Thermo Fisher Scientific/PA5- 15246

PI3KCA Antibody Thermo Fisher Scientific/PA5-18610

PI3KCA Antibody Thermo Fisher Scientific/PA5-27192

PI3KCA Antibody Thermo Fisher Scientific/MA5-17149

PIK3R1 Antibody Thermo Fisher Scientific/710400

PI3KR2 Antibody Thermo Fisher Scientific/PA5- 15262

PSMG1 Antibody Thermo Fisher Scientific/PA5-31298

PSMG1 Antibody Abgent/AI 12626

PSMG1 Antibody Acris/TA338919

PSMG1 Antibody Acris/TA338920

RAF1 Antibody Thermo Fisher Scientific/PA5-29333

RAF1 Antibody Thermo Fisher Scientific/PA5- 14941

RAF1 Antibody Thermo Fisher Scientific/MAl-90087

RAF1 Antibody Thermo Fisher Scientific/MA 1-21482 RAF1 Antibody Thermo Fisher Scientific/PA5 -20970

RAF1 Antibody Cell Signaling Technology /53745S

Ras- H Antibody Thermo Fisher Scientific/PA5- 14863

Ras- H Antibody Thermo Fisher Scientific/MAl-19726

Ras- H Antibody Thermo Fisher Scientific/PA5-22392

Ras- K Antibody Thermo Fisher Scientific/MAl-91046

Ras- K Antibody Thermo Fisher Scientific/415700

RASGRF1 Antibody Thermo Fisher Scientific/PA5- 14531

RASGRF1 Antibody Abcam/ab 190081

RASGRF2 Antibody Thermo Fisher Scientific/PA5 -28867

RASGRF2 Antibody Thermo Fisher Scientific/PA5-28857

RASGRF2 Antibody Abcam/ab 176008

Ras-N Antibody Thermo Fisher Scientific/PA5- 14833

RICTOR Antibody Thermo Fisher Scientific/PA5-14104

RPS6KB2 Antibody Thermo Fisher Scientific/PA5-26010

RPS6KB2 Antibody Thermo Fisher Scientific/PA5-15235

Raptor Antibody Thermo Fisher Scientific/PA5-20126

RSK1 (RPS6KA1) Antibody Thermo Fisher Scientific/LF-MA0183

RSK1 (RPS6KA1) Antibody Thermo Fisher Scientific/PA5-15135

RSK1 (RPS6KA1) Antibody Thermo Fisher Scientific/AH01472

RSK1 (RPS6KA1) Antibody Thermo Fisher Scientific/MA5-15341

RSK1 (RPS6KA1) Antibody Thermo Fisher Scientific/PA5-20134

RSK1 + RSK2 + RSK3 Antibody Thermo Fisher Scientific/PA5-17680

RSK1 + RSK2 + RSK3 Antibody Thermo Fisher Scientific/MA5-15040

RSK2 (RPS6KA3) Antibody Thermo Fisher Scientific/PA5 -29280

RSK2 (RPS6KA3) Antibody Thermo Fisher Scientific/PA5-15140

RSK2 (RPS6KA3) Antibody Thermo Fisher Scientific/MA5-15920

RSK3 (RPS6KA2) Antibody Thermo Fisher Scientific/PA5 -29222

SMAD4 Antibody Thermo Fisher Scientific/PA5-35330

SMAD4 Antibody Thermo Fisher Scientific/710714

SOS1 Antibody Thermo Fisher Scientific/MA5- 17234

SPRED1 Antibody Millipore/MABN478

SPRED2 Antibody Thermo Fisher Scientific/PA5-31038

SPRED2 Antibody Thermo Fisher Scientific/PA5 -20625

SPRED2 Antibody Abcam/ab50535

SPRED2 Antibody Abcam/ab58127

SPRY1 Antibody Thermo Fisher Scientific/PA5-43582

SPRY1 Antibody Abcam/ab75492

SPRY2 Antibody Thermo Fisher Scientific/PA5-43584

SPRY2 Antibody Thermo Fisher Scientific/PA5-43583

SPRY3 Antibody Abcam/abl80037

SPRY3 Antibody Novus Bio/AF5735

SPRY3 Antibody ProSci/30-623

SPRY4 Antibody Thermo Fisher Scientific/PA5-31401

SPRY4 Antibody Thermo Fisher Scientific/40-9300

STAT1 Antibody Thermo Fisher Scientific/MAl-037 STAT3 Antibody Thermo Fisher Scientific/710077

STAT3 Antibody Thermo Fisher Scientific/PAl -86605

STK11 Antibody Thermo Fisher Scientific/PA5- 14074

STK11 Antibody Thermo Fisher Scientific/PA5- 14072

STK11 Antibody Thermo Fisher Scientific/MA5- 14831

STK11 Antibody Thermo Fisher Scientific/PAl-12767

STK11 Antibody Thermo Fisher Scientific/PA5-20325

TP53 Antibody Thermo Fisher Scientific/PA5 -27822

TP53 Antibody Thermo Fisher Scientific/MAl-7629

TP53 Antibody Thermo Fisher Scientific/701270

TP53 Antibody Thermo Fisher Scientific/710294

Phospho-p38 MAPK (Thrl80/Tyrl82) Antibody Cell Signaling Technology /4511

Phospho-p38 MAPK (Thrl80/Tyrl82) Antibody Cell Signaling Technology /9211

Phospho-p38 MAPK (Thrl80/Tyrl82) Antibody Cell Signaling Technology /4631

Phospho-p38 MAPK (Thrl80/Tyrl82) Antibody Cell Signaling Technology/9215

Phospho-p38 MAPK (Thrl80/Tyrl82) Antibody Cell Signaling Technology/9216

Phospho-MEK3/MEK6 (Serl89, Ser207) Thermo Fisher Scientific/PA5- 17887

Antibody

Phospho-MKK3 (Serl89)/MKK6 (Ser207) Cell Signaling Technology/12280

Antibody

Phospho-MKK3 (Serl89)/MKK6 (Ser207) Cell Signaling Technology /9236

Antibody

Phospho-MSKl (Thr581) Antibody Cell Signaling Technology /9595

Phospho-MAPKAPK2 (Thr334) Antibody Cell Signaling Technology /3007

Phospho-MAPKAPK2 (Thr334) Antibody Thermo Fisher Scientific/PA5- 17791

Phospho-SEKl/MKK4 (Ser257) Antibody Cell Signaling Technology /4514

Phospho-SEKl/MKK4 (Ser257/Thr261) Cell Signaling Technology 19156

Antibody

[0069] The immunoprecipitated RAS -RAF -MAPK pathway proteins may be reduced and alkylated prior to fragmentation (e.g., digestion). Samples that have been reduced and alkylated may comprises modifications, such as to cysteine residues (e.g., CAM). Where a RAS -RAF -MAPK peptide of SEQ ID NO: 1 - 1400 shows modification resulting from, for example, reduction/alkylation, the non-modified peptide is also encompassed. For example, in each instance where a RAS-RAF-MAPK pathway peptide of SEQ ID NO: 1 - 1400 is referred to, also encompassed are unmodified peptides of SEQ ID NO: 1 - 1400.

[0070] The samples may optionally be desalted prior to analysis by mass spectrometry.

Both enzymatic and chemical digestion is encompassed. Enzymatic digestion includes, but is not limited to, digestion with a protease such as, for example, trypsin, chymotrypsin, AspN, GluC, LysC, LysN, ArgC, proteinase K, pepsin, Clostripain, Elastase, GluC biocarb, LysC/P, LysN Promise, Protein Endopeptidase, Staph Protease or thermolysin. Chemical digestion includes use of, for example, CNBr, iodosobenzoate and formic acid. [0071] In some embodiments, after fragmentation (e.g., digestion), peptide samples are analyzed by mass spectrometry (MS), and the resulting spectra are compared with theoretical spectra from known proteins to determine the peptides and proteins in a sample. For RAS- RAF-MAPK pathway proteins, discovery MS is cumbersome and time consuming and is not a viable clinical method. Therefore, the inventors have identified novel peptides that associate with RAS-RAF-MAPK pathway proteins for use in the IP-MS methods. Use of these peptides in targeted MS, and IP -targeted MS methods allows quantitation of even low abundant RAS- RAF-MAPK proteins. Moreover, use of these peptides in targeted MS, and in IP-targeted MS methods, allows quantitation of phosphorylated RAS-RAF-MAPK proteins.

[0072] Theoretically, peptides useful in MS to detect and quantify RAS-RAF-MAPK pathway proteins can be designed by use of computer software and the like. Surprisingly, however, many of these potential peptide sequences are unsuitable or ineffective for use in MS-based assays, including SRM/MRM and PRM. Because it was not possible to predict the most suitable peptides for MS analysis, it was necessary to experimentally identify modified and unmodified peptides to develop as clinical reagents. To complicate the analysis, it was discovered that certain peptides useful when assaying typical samples were not predictive when assaying samples that had undergone immunoprecipitation.

[0073] Typically, targeted MS is performed by quantifying specific unique peptides of the protein. In some embodiments, known amounts of isotope-labeled (e.g., heavy isotope- labeled) versions of these targeted peptides can be used as internal standards for absolute quantitation. In some instances, proteins of interest are not detectable even after identifying unique peptide standards. The combination of specific antibodies with specific target peptides has allowed the inventors to improve the sensitivity of detection of RAS-RAF-MAPK pathway proteins by MS, and has allowed for lower levels of detection and lower levels of

quantification than ever previously seen. See, e.g., Figure 3.

[0074] In some embodiments, the RAS-RAF-MAPK pathway peptides provided in the kits, and useful in the described methods, are listed in Table 3. SEQ ID Nos: 1-700 are native peptide sequences useful in identifying the RAS-RAF-MAPK pathway proteins recited in the "Target ID" column. Certain peptide sequences are phosphorylated at certain residues as shown in parentheses "(P03H2)" following the modified residue.

[0075] Certain peptides are modified at cysteine residues as shown by "(CAM)" following the modified residue. The "CAM" post-translational modification is well known to those of skill in the art to mean carbamidomethylation, resulting from alkylation of the protein/peptide. The peptides may be as shown in Table 3, or may be non-modified version of these peptides lacking carbamidomethylation. Table 3. List of peptides to quantitate RAS-RAF-MAPK Pathway proteins

[0076] In some embodiments, the peptides reagents, and the peptides useful in the methods, are recited in Table 5 (SEQ ID Nos: 13, 15, 18, 28, 31, 47, 48, 51, 127, 128, 132, 133, 141, 142, 143, 144, 171, 173, 177, 180, 181, 182, 183, 190, 192, 213, 219, 220, 224, 226, 231, 233, 234, 235, 236, 258, 270, 295, 297, 300, 301, 302, 304, 306, 308, 310, 347, 348, 353, 362, 392, 393, 405, 416, 429, 443, 446, 451, 467, 483, 487, 501, 502, 506, 507, 510, 512, 521, 522, 523, 528, 572, 580, 588, 591, 598, 603, 615, 617, 635, 636, 655, 657, 660, 662, 670, 678, 683, 691, 692, 696, 698, and 699). In some embodiments, the peptides of Table 5 are useful in multi-plex MS methods.

[0077] In some embodiments, protein samples are denatured or solubilized before fragmentation.

[0078] In some embodiments, the fragmentation protocol uses chemical cleavage. In some embodiments, the chemical cleavage uses CNBr. In some embodiments, the

fragmentation protocol is done using an enzyme. In some embodiments, the fragmentation protocol uses MS-grade commercially available proteases. Examples of proteases that may be used to digest samples include trypsin, endoproteinase GluC, endoproteinase ArgC, pepsin, chymotrypsin, LysN protease, LysC protease, GluC protease, AspN protease, proteinase K, and thermolysin. In some embodiments, a mixture of different proteases are used and the individual results are combined together after the digestion and analysis. In some

embodiments, the digestion is incomplete in order to see larger, overlapping peptides. In some embodiments, the antibody digestion is performed with IdeS, IdeZ, pepsin, or papain to generate large antibody domains for "middle-down" protein characterization. In some embodiments, the fragmentation protocol uses trypsin that is modified. In some embodiments, a protein: protease ratio (w/w) of 10: 1, 20: 1, 25: 1, 50: 1, 66: 1, or 100: 1 may be used. In some embodiments, the trypsin used is at a concentration of about 100 ng/ml-1 mg/ml, or about 100 ng/ml-500μg/ml, or about 100 ng/ml-100μg/ml, or about 1 μg/ml-lmg/ml, or about 1 μg/ml-500μg/ml, or about 1 μg/ml-100μg/ml, or about 10 μg/mg-lmg/ml, or about 10 μg/mg- 500 μg/ml, or about 10 μg/mg-100μg/ml. In some embodiments, the digestion step is for about 10 minutes to 48 hours, or about 30 minutes to 48 hours, or about 30 minutes to 24 hours, or about 30 minutes to 16 hours, or about 1 hour to 48 hours, or about 1 hour to 24 hours, or about 1 hour to 16 hours, or about 1 to 8 hours, or about 1 to 6 hours, or about 1 to 4 hours. In some embodiments, the digestion step is incubated at a temperature from about 20°C to 45°C, or from about 20°C to 40°C, or from about 22°C to 40°C, or from about 25°C to 37°C. In some embodiments, the digestion step is incubated at about 37°C or 30°C. In some embodiments, a step is included to end the digestion step. The step to end the digestion protocol may be addition of a stop solution or a step of spinning or pelleting of a sample. In some embodiments, the digestion is followed by guanidination.

[0079] In some embodiments, the fragmentation protocol includes use of protein gels.

In some embodiments, the fragmentation protocol comprises in-gel digestion. An exemplary commercially available kit for performing in-gel digestion is the In-Gel Tryptic Digestion Kit (Thermo Fisher Cat#89871).

[0080] In some embodiments, the fragmentation protocol is carried out in solution. An exemplary commercially available kit for performing in-solution digestion is the In-Solution Tryptic Digestion and Guanidination Kit (Thermo Fisher Cat#89895).

[0081] In some embodiments, the fragmentation protocol uses beads. In some embodiments, the fragmentation protocol comprises on-bead digestion. In some embodiments, agarose beads or Protein G beads are used. In some embodiments, magnetic beads are used.

[0082] In some embodiments, protein samples are separated using liquid

chromatography before MS analysis. In some embodiments, fragmented samples are separated using liquid chromatography before MS analysis.

[0083] The IP and IP-MS methods described herein are capable of detecting phosphorylated RAS-RAF-MAPK pathway proteins, including those described in Table 4.

Table 4. List of Total RAS-RAF-MAPK Pathway Target Proteins 3 BRAF P15056 42 MAPK3 P27361

4 CALM1 P62158 43 MAPK8 P45983

5 CAMK2A Q9UQM7 44 MAPK9 P45984

6 CCND1 P24385 45 MET P08581

7 CDH1 P12830 46 NF1 P21359

8 CDH2 P 19022 47 NOTCH 1 P46531

9 CDK2 P24941 48 NRAS P01111

10 CDKN1A P38936 49 PAK1 013153

11 CDKN1B P46527 50 PARP1 P09874

12 CDKN2A Q8N726 51 PGR P06401

13 CTNNA1 P35221 52 PIK3CA P42336

14 CTNNB1 P35222 53 PIK3R1 P27986

15 DDR2 Q16832 54 PIK3R2 000459

16 DUSP22 Q9NRW4 55 PSMG1 095456

17 DUSP4 Q13115 56 RAF1 P04049

18 DUSP6 Q 16828 57 RASA1 P20936

19 EGFR P00533 58 RASGRF1 Q13972

20 EIF2A Q9BY44 59 RASGRF2 014827

21 EIF4E P06730 60 RICTOR Q6R327

22 EIF4EBP1 Q13541 61 RPS6KA1 Q15418

23 ERBB2 P04626 62 RPS6KA2 Q15349

24 ERBB3 P21860 63 RPS6KA3 P51812

25 ERBB4 Q15303 64 RPS6KB2 Q9UBS0

26 ESR1 P03372 65 RPTOR Q8N122

27 FBX07 Q9Y3I1 66 SMAD3 P84022

28 FGFR1 P11362 67 SMAD4 Q13485

29 FGFR3 P22607 68 SOS1 Q07889

30 FOS P01100 69 SPRED1 Q7Z699

31 FOXOl 012778 70 SPRED2 Q7Z698

32 FOX03 043524 71 SPRY1 043609

33 HRAS P01112 72 SPRY2 043597

34 IQGAP1 P46940 73 SPRY3 043610

35 JAK1 P23458 74 SPRY4 Q9C004

36 JAK2 060674 75 STAT1 P42224

37 KRAS P01116 76 STAT3 P40763

38 MAP2K1 Q02750 77 STK11 Q15831

39 MAP2K2 P36507 78 TP53 P04637

79 ATF2 P15336 80 MAPK12 P53778

81 MAPK13 015264 82 MAPK14 Q16539

83 MAP2K3 P46734 84 MAP2K6 P52564

85 RPS6KA5 075582 86 MAPKAPK2 P49137

[0084] In some embodiments, the RAS-RAF-MAPK pathway peptides used in the MS methods described herein have limits of detection considered useful in clinical and research methods. See, e.g, Table 5. In some embodiments, the RAS-RAF-MAPK pathway peptides used in the MS and IP-MS methods comprise or consist of the peptides described in Table 5. In some embodiments, the peptides of Table 5 are detectably labelled.

Table 5. Lower Limit of uantitation of Pe tides for RAS-RAF-MAPK Pathwa Proteins

SPRY1 4 FirC(CAM)lEQrC(CAM)lGK 657 0.14

SPRY2 2 LHGLPEHR 660 3.70

SPRY2 4 EDLGLHAYR 662 0.05

SPRY4 2 TSHVENDYIDNPSLALTTGPK 670 3.70

STAT3 1 FLQESNVLYQHNLR 678 3.70

STAT3 6 FNILGTNTK 683 0.05

TP53 1 TYQGSYGFR 691 0.05

TP53 2 LGFLHSGTAK 692 1.23

TP53 6 VEYLDDR 696 0.14

TP53 8 ALPNNTSSSPQPK 698 0.14

TP53 9 ELNEALELK 699 0.14

Table 6. List of antibodies for multi- lex IP sin le- lex IP +/- MS .

FBX07 Millipore ABN1038

FBX07 Novus Bio NBP1-55051

FOS Thermo Fisher Scientific MA5-15055

FOS Thermo Fisher Scientific PA5- 17489

FOXOl Thermo Fisher Scientific MA5- 14846

FOX03 Cell Signaling Technology 12829

HRAS Thermo Fisher Scientific 33-7200

KRAS/HRAS/NRAS Thermo Fisher Scientific MA1-012

IQGAP1 Thermo Fisher Scientific 33-8900

JAK1 Cell Signaling Technology 3332

JAK1 Cell Signaling Technology 3344

JAK2 Cell Signaling Technology 3230

MAP2K1 Thermo Fisher Scientific MA5-15093

MAP2K2 Abeam ab28834

MAPK1 Thermo Fisher Scientific PA1-21016

MAPK1/MAPK3 Proteintech 66192-1-Ig

MAPK11 Abeam ab80116 pan MAPK8/9 Proteintech 51153-1-AP

MAPK9 Thermo Fisher Scientific PA5-28664

MET Thermo Fisher Scientific PA1-28026

MET Thermo Fisher Scientific PA5-17148

MTOR Thermo Fisher Scientific PA1-188

MTOR Thermo Fisher Scientific PA1-518

NF1 Millipore 07-730

NF1 Abeam ab 17963

NOTCH 1 Cell Signaling Technology 3608

PAK1 Thermo Fisher Scientific 71-9300

Pan-Cadherin Thermo Fisher Scientific 71-7100

Pan-Cadherin Thermo Fisher Scientific PA1-37199

PARP1 Thermo Fisher Scientific MA5-15031

PIK3CA Millipore 09-481

PIK3R2 Thermo Fisher Scientific MA1-90048

PSMG1 Cell Signaling Technology 13378

RAF1 Millipore 07-396

RAF1 Thermo Fisher Scientific PA5-28652

RASA1 Abeam ab2922

RASA1 Thermo Fisher Scientific MA4-001

RICTOR Cell Signaling Technology 9476

RICTOR Abeam ab70373

RSK1 (RPS6KA1) Cell Signaling Technology 8408

RSK1 (RPS6KA1) Thermo Fisher Scientific PA5-21699

RSK2 (RPS6KA3) Thermo Fisher Scientific 38-6800

[0085] In one instance, total TP53 and phosphorylated TP53 are evaluated together. In some embodiments, KRAS, HRAS, and NRAS are evaluated together. In other embodiments, EGFR, SOSl, ARAF, BRAF, RAFl, KRAS, HRAS, and NRAS, RPS6KA1, PIK3R1, PIK3R2 and PIK3CA are evaluated together. In some embodiments, ERBB2, SOSl, ARAF, BRAF, RAFl, KRAS, HRAS, and NRAS, RPS6KA1, PIK3R1, PIK3R2 and PIK3CA are evaluated together. In some embodiments, SPRY1, SPRY2, DUSP4, RASA1, NF1, SPRED1, SPRED2, KRAS, HRAS, and NRAS are evaluated together. In some embodiments, MAPKl/3 and TP53 are evaluated together (including either total protein or phosphorylated forms, such as, for example MAPKl/3 (including phosphorylated form pThr202/pTyr204) and TP53 (including phosphorylated form pSerl5)). In some embodiments, PIK3R1, PIK3R2, PIK3CA, ARAF, BRAF, RAFl, KRAS, HRAS, NRAS, RPS6KA1, RPS6KB1, AKT1, AKT2, AKT3, MTOR, phosphor-TP53 (pSerl5), phosphor-MAPKl/3 (pThr202/pTyr204) and phospho-MAPK8/9 (pThrl83/pTyrl85) are evaluated together. Any of the antibodies referenced in Table 6 corresponding to these proteins may be employed in a multi-plex IP format, for example, as in the groups set forth in Table 7.

Table 7. Selected combinations of antibodies for multi-plex IP (+/- MS) (when more than one

EXAMPLES

[0086] The following examples are provided to illustrate certain disclosed

embodiments and are not to be construed as limiting the scope of this disclosure in any way.

Example 1 - Immunoprecipitation of RAS-RAF-MAPK pathway proteins and Discovery- MS

[0087] RAS-RAF-MAPK pathway proteins play central roles in diseases including cancer. The identification of RAS-RAF-MAPK pathway proteins, while desired as a means for monitoring disease progression, and as a tool for scientific research, has been limited in part because of the low abundance of RAS-RAF-MAPK pathway proteins, and in part due to a lack of validated methods and reagents. Phosphorylated RAS-RAF-MAPK pathway proteins are particularly important to identify and quantify as a measure of protein activation status, and also as markers for disease progression. As shown in Figure 1, methods and reagents for detecting RAS-RAF-MAPK pathway proteins, including phosphorylated proteins, and their protein interactions, were designed and tested. Multiplex immunoprecipitation (IP) to MS (mlP-MS) was assessed for the ability to measure total and phosphorylated RAS-RAF-MAPK pathway targets. mlP-MS methods were also compared to existing singleplex immunoassay (Western Blot (WB) and ELISA) and multiplex Luminex assays.

[0088] Immunoprecipitation and MS Sample Preparation

[0089] The Thermo Scientific™ Pierce MS-Compatible Magnetic IP Kit (Protein A/G) was used to screen and validate antibodies for 78 RAS-RAF-MAPK pathway proteins from 500μg of cell lysate. Validated antibodies were biotinylated with the Thermo Scientific™ Pierce Antibody Biotinylation Kit for IP. The Thermo Scientific™ Pierce MS-Compatible Magnetic IP Kit (Streptavidin) was used to perform the single or multiplex IPs for target enrichment. IP samples were processed by an in-solution digestion method where IP eluates were reconstituted in 6M Urea, 50mM triethylammonium bicarbonate (TEAB), pH 8.5 followed by reduction, alkylation and trypsin digestion overnight at 37°C. The digested samples were acidified with trifluoroacetic acid (TFA) before MS analysis.

[0090] Immunoprecipitation with Enzyme Elution and MS Sample Preparation

[0091] An additional method employing a shorter trypsin digestion was developed

(data not shown). Antibodies and appropriate lysate were incubated overnight at 4°C with rotation. The next day, immune complexes were washed and allowed to incubate with l ^g trypsin in 50mM TEAB for 1 hour and 37°C with 800rpm shaking in a thermomixer. Enzyme derived IP elutions were separated from the beads and the beads were allowed to incubate with an MS compatible elution buffer (Thermo Fisher Scientific, PN: 90409) for 10 minutes at room temperature. This second elution was collected separately. The second elution was dried down in a speedvac and processed with a urea based in-solution digestion method in which IP eluates were reconstituted in 6M Urea, 50mM TEAB, pH 8.5 followed by reduction, alkylation and trypsin digestion overnight at 37°C. The digested samples were acidified with TFA before MS analysis.

[0092] The enzyme-derived IP elution was combined with 25ng Green Fluorescent

Protein (GFP) and exposed to a solution of Tris[2-

Carboxyethyl]phosphine (TCEP)/Chloroacetamide at a 1 :4 ratio for 5-10 minutes at 95°C and then allowed to cool briefly before addition of 600ng trypsin and incubation for 2 hours at 37°C with 500rpm in a thermomixer. The trypsin-digested samples were neutralized with 10% TFA and dried down in a speedvac. Samples were reconstituted in 5% acetonitrile (ACN) and 0.1% TFA and taken to LC-MS analysis.

[0093] Liquid Chromatography and Mass Spectrometry

[0094] Prior to MS analysis, tryptic digest samples were desalted on-line using the

Thermo Scientific™ Acclaim™ PepMap 100 C18 Trap Column. For discovery MS, the samples were analyzed by nanoLC-MS/MS using a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano System and Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole- Orbitrap Mass Spectrometer. For targeted MS, the samples were analyzed using the UltiMate 3000 RSLCnano System and the Thermo Scientific™ TSQ™ Vantage™ Mass Spectrometer (SRM mode) or the Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (PRM mode).

[0095] MS Data Analysis

[0096] Discovery MS data were analyzed with Thermo Scientific™ Proteome

Discoverer™ 1.4 to assess percent sequence coverage, unique peptides, MSI intensities, spectral counts and PTMs. The Proteome Discoverer software searches were executed using the Uniprot human protein database. Tryptic peptides with highest MS 1 intensity and relevant phosphorylation sites were selected from the discovery data for targeted assay development. For targeted MS data analysis, Thermo Scientific™ Pinpoint software and Skyline software (University of Washington) were used to measure limit of quantitation (LOQ) from the calibration curve and target analyte concentration from unknown samples.

[0097] Results

[0098] As shown in Figure 2, RAS-RAF-MAPK pathway proteins were

immunoprecipitated from HCT116 or BT549 cell lysates with Thermo Scientific™ Pierce MS- Compatible Magnetic IP Kits (Protein A/G or Streptavidin) for MS analysis. A variety of antibodies were screened to determine effectiveness in both ability to IP RAS-RAF-MAPK pathway proteins, and also for their usefulness when combined with MS. Table 1 (above) provides a list of antibodies validated for use in the IP-MS methods. Table 2 (above) provides a list of antibodies tested, but found to be less successful.

[0099] Higher numbers of unique peptides were identified in IP enriched samples as compared to neat (non-IP-enriched) lysate. See Figure 2. Protein isoforms and interacting protein partners were identified for RAS (K, H, N), JNKs (MAPK8, MAPK9), PI3K (PIK3CA, PIK3CB, PIK3R1, PIK3R2), and SOS l targets. Relevant phosphor peptides were identified for ERbb2, PIK3R2, RAF1, RPS6KA1, SOSl and TP53 targets. Candidate quantitative peptides were selected for targeted MS assay development.

[00100] Limits of detection (LOD) and lower limits of quantification (LLOQ) were analyzed for RAS-RAF-MAPK pathway proteins, including BRAF, ERBB2, FOS, FOXOl, FOX03, HRAS, KRAS, MAP2K1, MAP2K2, MAPK1, MAPK3, MAPK8, MAPK9, NRAS, RPS6KA1, SOSl, and TP53. Results are presented in Figure 3. The assay dynamic range, representing the concentration range between the lower to upper limits of quantification (LLOQ to ULOQ), is the range where protein concentration is measurable with acceptable levels of accuracy and precision. To ensure linearity of the measurement, for each internal standard peptide the linear signal-to-abundance range (LLOQ and ULOQ) was determined from dilution series experiments spanning concentrations of 300-0.05 fmol on column, spiked into a constant light peptide at 36fmol and 200ng of equimolar concentration of 6 proteins digest. In some instances, lower limits of detection and quantification may be established, even though these lower limits were not presently tested. Thus, the lower limits are no more than the numbers determined experimentally.

Example 2 - Multiplex IP of RAS-RAF-MAPK pathway proteins and multiplex MS

[00101] Multiple RAS-RAF-MAPK pathway protein targets were enriched

simultaneously from unstimulated and human insulin-like growth factor (hlGF-l)-stimulated HCT116 lysate with biotinylated antibodies and Thermo Scientific™ Pierce MS-Compatible Magnetic IP Kit (Streptavidin). HCT116 cells were starved in 0.1% charcoal stripped fetal bovine serum (FBS) for 24 hours before stimulation with lOOng/ml of IGF for 15 minutes. Validated IP-MS antibodies are biotinylated for 29 RAS-RAF-MAPK pathway targets using the Thermo Scientific™ Pierce Antibody Biotinylation Kit for IP (PN: 90407) as

recommended in instruction manual. 29 total targets were grouped into three different panels for multi-plex IP. ^g of each biotinylated antibody for each panel comprising 10-15 total targets were added simultaneously to 1000μg of control and IGF stimulated HCT116 cell lysate in duplicate. IP was performed as recommended in the Thermo Scientific™ Pierce MS- Compatible Magnetic IP Kit (Streptavidin) (PN: 90408) with the following modification. 5 micrograms of streptavidin magnetic beads per microgram of biotinylated antibody concentration was used for multi-plex IP.

[00102] IP samples were processed by an in-solution digestion method where IP eluates were reconstituted in 6M Urea, 50mM TEAB, pH 8.5 followed by reduction (5mM TCEP for 30 minutes at 35°C), alkylation (20mM Iodoacetamide in dark at room temperature for 30 minutes) and trypsin digestion overnight at 37°C. The digested samples were acidified with 3.5μ1. of 10% TFA before discovery MS analysis. For discovery MS, the samples were analyzed by nanoLC-MS/MS using a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano System and Thermo Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer. Briefly, the digested samples were cleaned on-line using the C18 trap column (Thermo Fisher Scientific, PN: 164564) followed by reversed-phase separation using the analytical CI 8 column (75 μιτι i.d. x 15 cm, nanoViper, 3μιη particle size, Thermo Fisher Scientific, PN: ES800) with a 2-30% gradient of Buffer B using Buffer A (0.1% formic acid) and Buffer B (0.1% formic acid/99.9 % acetonitrile) at 0.300 μΙ7ιηίη.

[00103] Figure 4 shows that the IP-nanoLC-MS/MS analysis was able to identify 14 proteins in the multiplex assay panel 1. MS analysis of multiplex assay identified interacting proteins (KRAS-2b, MAPK8, and SOS1) of RAS-RAF-MAPK Pathway Targets. Table 6 provides listings of the antibodies used in this multiplex IP.

[00104] Figure 5 shows that the IP-nanoLC-MS/MS analysis was able to identify 12 proteins in the multiplex assay panel 2. Table 6 provides listings of the antibodies used in this multiplex IP.

[00105] Figure 6 shows that the IP-nanoLC-MS/MS analysis was able to identify 15 proteins in the multiplex assay panel 3. MS analysis of multiplex assay identified interacting proteins (PIK3CA, PIK3CB, and PIK3R1) of RAS-RAF-MAPK Pathway Targets. Table 6 provides listings of the antibodies used in this multiplex IP.

[00106] A summary of RAS-RAF-MAPK pathway proteins that were identified and quantified using the IP-MS methods described herein is provided in Figure 7. Most of the RAS-RAF-MAPK pathway targets were not identified in discovery MS and quantitated by targeted MS (PRM or SRM) without enrichment by immunoprecipitation.

[00107] Immunoprecipitation using particular selected antibodies resulted in a higher yield of RAS-RAF-MAPK pathway target proteins and less non-specific binding proteins than MS alone. IP-MS assay was also more successful than other commercially available non-MS assays. Furthermore, IP to MS analysis of the RAS-RAF-MAPK pathway proteins enabled identification of multiple isoforms, relevant protein interactions and phosphorylation sites. Three panels of mlP-tMS assays allowed simultaneous quantitation of 14, 12 and 15 RAS- RAF-MAPK pathway proteins in the low to sub-fmol range from unstimulated and IGF stimulated HCT116 cell lysates. Major advantages of the MS-based assay are high confidence in target identity coupled with simultaneous quantitation of multiple targets, interacting proteins and their phosphophorylated forms.