Compositions and methods for determining protein interactions

Abstract

Embodiments of the disclosure may comprise a photo-dependent proximity labeling system comprising an activatable probe and a retrievable substrate. The activatable probe may be, for example, an integrin binding peptide operatively coupled to a lumichrome molecule, and the retrievable substrate may be, for example, a biotin phenol. In some aspects, the activatable probe is activatable by light, thereby inducing labeling of adjacent molecules with a retrievable substrate. Also disclosed are methods for labeling proteins, identifying protein interactions or binding partners, and methods for identifying a therapeutic target in an individual using a composition of the disclosure.

Description

COMPOSITIONS AND METHODS FOR DETERMINING PROTEIN INTERACTIONS

[0001] This application claims priority of U.S. Provisional Application Nos. 63 / 540503 filed September 26, 2023 and 63 / 691118 filed September 5, 2024, both of which are hereby incorporated by reference in their entirety.BACKGROUND OF THE INVENTION

[0002] This invention was made with government support under GM145852 awarded by the National Institutes of Health. The government has certain rights in the invention.I. Field of the Invention

[0003] This invention relates to the field of proteomics, biomarkers, and medicine.SUMMARY OF THE INVENTION

[0004] Various aspects of this disclosure relate at least in part to the discovery of chemical probes capable of binding to a protein of interest, where the probes comprise photosensitizer moieties, such as lumichrome, that can convert reactive substrates, such as electron rich aromatic compounds, to chemically label binding partners or other proteins that are in physical proximity to the protein of interest at a given time. The photosensitizer moiety can be a molecule capable of catalyzing a conversion of the reactive substrate when the photosensitizer moiety is activated by exposure to light. In various aspects, the photosensitizer moiety can photo-oxidize certain reactive substrates when activated by exposure to light. The reactive substrates, when exposed to the activated photosensitizer moiety, can react with one or more proteins, including proteins within a defined proximity of the exposure to the activated photosensitizer. In various aspects, the reactive substrate is a retrievable substrate. The retrievable substrate can have a retrievable moiety, such as biotin, linked to the reactive substrate. According to various aspects, when the retrievable substrate reacts with the one or more proteins, the retrievable moiety is attached to the one or more proteins, which can allow for the one or more proteins to be tagged for retrieval. For example, in various aspects, the retrievable substrate is photo-oxidized by the photosensitizer and covalently attaches a molecule comprising biotin to the one or more proteins.

[0005] Some aspects of the disclosure relate at least in part to a photo-dependent proximity labeling system comprising an activatable probe and a retrievable substrate. In some aspects,the activatable probe and the retrievable substrate are different molecules. In various aspects, the photosensitizer is attached to a ligand capable of binding to a protein of interest. The ligand can be any ligand, including but not limited to a small molecule, a small peptide, a protein, or any molecule capable of binding a protein of interest. In some aspects, the ligand comprises, consists of, or consists essentially of a peptide, small molecule, or biologic. In some aspects, the ligand comprises, consists of, or consists essentially of a protein or peptide that binds an integrin and / or an integrin complex. In some aspects, the ligand comprises, consists of, or consists essentially of an Arg-Gly-Asp peptide, or a functional derivative thereof. In some aspects, a retrievable substrate comprises, consists of, or consists essentially of a structure ofBP, AP, SI, S2, S3 or S4:Si bslrate 3 (33) Substrate 4 (S4)

[0006] In various aspects, a retrievable substrate comprises, consists of, or consists essentially of a structure of BP. In various aspects, a retrievable substrate comprises, consists of, or consists essentially of a structure of AP. In various aspects, a retrievable substrate comprises, consists of, or consists essentially of a structure of SI. In various aspects, a retrievable substrate comprises, consists of, or consists essentially of a structure of S2. In various aspects, a retrievable substrate comprises, consists of, or consists essentially of a structure of S3. In various aspects, a retrievable substrate comprises, consists of, or consistsessentially of a structure of S4. In various aspects, a retrievable substrate comprises, consists of, or consists essentially of a biotin linked to a phenol derivative, a diazirine derivative, a benzophenone derivative, an amine derivative, or an aryl azide derivative. In various aspects, a retrievable substrate comprises, consists of, or consists essentially of a biotin linked to an phenol-derivative. In various aspects, a retrievable substrate comprises, consists of, or consists essentially of a biotin linked to a diazirine. In various aspects, a retrievable substrate comprises, consists of, or consists essentially of biotin phenol.

[0007] In various aspects, a photosensitizer comprises, consists essentially of, or consists of a molecule having the formula of Formula (I). In some aspects, the photosensitizer attached to the ligand comprises, consists essentially of, or consists of a molecule having the formula of Formula (I):Formula I.

[0008] In various aspects, the photosensitizer and / or photoactivatable probe is activatable by light. In various aspects, the photosensitizer and / or photoactivatable probe is activatable by light of a wavelength of 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 nm, or any range derivable therein. In various aspects, the photosensitizer and / or photoactivatable probeis activatable by light of a wavelength of 440 nm. In various aspects, the photosensitizer and / or photoactivatable probe is activatable by light comprising wavelengths of 365 nm and / or 440 nm. In some aspects, a photosensitizer and / or photoactivatable probe activates a phenol-based reactive substrate when exposed to light comprising wavelengths of 365 nm and / or 440 nm. In some aspects, a photosensitizer and / or photoactivatable probe activates a diazirine-based reactive substrate when exposed to light comprising a wavelength of 440 nm.

[0009] According to various aspects, photo-dependent proximity labeling systems comprise a probe and a retrievable substrate, wherein the probe has a photosensitizing moiety capable of reacting with the retrievable substrate upon exposure to light. Any of the systems disclosed herein can be comprised in a delivery buffer, solution, or other carrier for use in the various methods disclosed herein.

[0010] Disclosed herein are methods of determining protein binding complexes, methods of determining protein binding partners of a protein of interest, methods of labeling binding partners of a ligand-binding molecule, methods of determining a therapeutic target, and methods of analyzing a sample from a patient. In various aspects, method of determining a therapeutic target are used to determine protein binding partners for a particular protein of interest. As a non-limiting example, the method can determine protein binding partners of one or more integrins in a sample, which can be used to determine therapeutic targets to be used against such protein binding partners. In various aspects, the methods are performed on a sample from a patient. The patient can have, be suspected of having, or be diagnosed with having a disease, such as but not limited to cancer, an autoimmune disease, an infection, a genetic disease, or a combination thereof. The sample can be from any tissue from the patient. In various aspects, the sample is from one or more tissues from the patient. The samples can be kept in a way that allows proteins within the sample to maintain protein binding activity.

[0011] In some aspects, the method comprises, consists of, or consists essentially of labeling a protein complex in a sample by introducing a photo-dependent proximity labeling system of the disclosure to the sample and exposing the sample to a wavelength of light capable of activating the activatable probe. In some aspects, the method comprises, consists of, or consists essentially of labeling a protein or protein complex in a sample, the method comprising: introducing an activatable probe having a structure of Formula (I) and a retrievable substrate to the sample; exposing the sample to a wavelength of light capable of activating the structure of Formula (I) thereby generating a set of proteins labeled with the retrievable substrate; retrieving the set of labeled proteins to generate a set of retrieved proteins; and analyzing the set of retrieved proteins. In some aspects, the retrievable substrate comprises,consists of, or consists essentially of a phenol-based or diazirine-based retrievable substrate. In some aspects, the retrievable substrate comprises, consists of, or consists essentially of phenol or diazirine bound to a retrievable moiety (e.g., biotin). In some aspects, the retrievable substrate comprises, consists of, or consists essentially of biotin phenol or biotin diazirine. In some aspects, the retrievable substrate comprises, consists of, or consists essentially of a structure of BP. In some aspects, retrieving the set of labeled proteins comprises, consists of, or consists essentially of streptavidin enrichment. In some aspects, the method comprises, consists of, or consists essentially of identifying one or more therapeutic targets in a patient, the method comprising: introducing a photo-dependent proximity labeling system of the disclosure to a biological sample from the patient, wherein the activatable probe binds to a protein of interest; and exposing the sample to a wavelength of light capable of activating the activatable probe to generate a set of labeled proteins. In some aspects, introducing a photodependent proximity labeling system of the disclosure comprises the steps of: introducing an activatable probe and a retrievable substrate prior to exposing the sample to light. In some aspects, introducing a photo-dependent proximity labeling system of the disclosure comprises the sequential steps of: introducing an activatable probe, introducing a retrievable substrate, and exposing the sample to light. In some aspects, the method comprises, consists of, or consists essentially of diagnosing cancer in a patient, said method comprising: introducing a photo-dependent proximity labeling system of the disclosure to a biological sample obtained from the patient, wherein the activatable probe binds to a protein of interest; exposing the sample to a wavelength of light capable of activating the activatable probe to generate a set of labeled proteins; and determining the identity of one or more proteins in the set of labeled proteins. In some aspects, the method comprises, consists of, or consists essentially of identifying binding partners of a protein of interest in a biological sample obtained from a patient, said method comprising: introducing a photo-dependent proximity labeling system of the disclosure to the biological sample wherein the activatable probe binds to the protein of interest; and exposing the sample to a wavelength of light capable of activating the activatable probe to generate a set of labeled proteins.

[0012] In some aspects, the protein complex comprises, consists of, or consists essentially of an integrin. In some aspects, the sample comprises, consists of, or consist essentially of a population of cells. The population of cells may be mammalian cells, such as human cells. In some aspects, the sample is a biological sample from a patient. In some aspects, the biological sample comprises, consists of, or consist essentially of a fresh sample, a frozen sample, and / or a fixed sample. In some aspects, the biological sample is not frozen and / or not fixed. In someaspects, the sample is a biopsy sample. In some aspects, the sample is from an individual having, suspected of having, or diagnosed with having cancer. In some aspects, the cancer originated in tissue from bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus in the patient. In some aspects, the cancer is a solid cancer. In some aspects, the cancer is a metastatic cancer. In some aspects, the biological sample comprises tissue having a size of approximately 300-500 mm3.

[0013] In some aspects, a photo-dependent proximity labeling system, activatable probe, and / or retrievable substrate is introduced at a concentration of at least, at most, approximately, or exactly 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109^ 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 pM, or any range derivable therein. In some aspects, a photo-dependent proximity labeling system comprises a probe introduced at a concentration of at least, at most, approximately, or exactly 50 pM. In some aspects, a photo-dependent proximity labeling system comprises a probe introduced at a concentration of at least, at most, approximately, or exactly 5-10 pM. In some aspects, a photo-dependent proximity labeling system comprises a retrievable substrate introduced at a concentration of at least, at most, approximately, or exactly 250 pM. In some aspects, a retrievable substrate comprises a biotin phenol. In some aspects, a biotin phenol is introduced at a concentration of at least, at most, approximately, or exactly 250 pM. In some aspects, the biotin phenol comprises a formula of BP. In some aspects, a molecule having a structure of Formula (I) is introduced at a concentration of at least, at most, approximately, or exactly 50 pM. In some aspects, a molecule having a structure of Formula (I) is introduced at a concentration of at least, at most, approximately, or exactly 5-10 pM.

[0014] In some aspects, a photo-dependent proximity labeling system is activated by exposure to light. In some aspects, the light comprises, consists of, or consists essentially of a wavelength of light is 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 nm, or any range derivable therein. In some aspects, the wavelength is 440 nm. In some aspects, the wavelength is 365 nm. In some aspects, a photo-dependent proximity labeling system, its components, and / or sample are exposed to light for between about 1, 2, 3, 4, or 5 minutes, or any range derivable thereof.

[0015] In some aspects, methods of the disclosure relate, at least in part, to retrieving proteins labeled with the retrievable substrate from the sample. In some aspects, retrieving the labeled proteins comprises, consists of, or consists essentially of streptavidin enrichment. In some aspects, the method further comprises, consists of, or consists essentially of analyzingthe sample to determine an identity of proteins labeled with the retrievable substrate. In some aspects, analyzing the sample comprises, consists of, or consists essentially of performing mass spectrometry, chromatography, electrophoresis, and / or a suitable protein identification assay. In some aspects, the labeled protein(s) are enriched at least about 2-fold. In some aspects, the labeled protein(s) comprises, consists of, or consists essentially of a proximity signature derivable from any one of Figures 5C-5G, Figures 4A-4D, and / or Figures 3A-3F. In some aspects, the labeled protein(s) or a proximity signature comprises, consists of, or consists essentially of ITGB4-isoform 2, CD55, MELTF, NECTIN2, DLG1, PTPRK, CSF2RA, FRMD6, VASN, FCGRT, PCDHB13, CA12, HRH1, SLC3A2-1, VCAM-1, EPHB3, EPHA4, SLIT2, FAP, CD44, TGFB1, CTNNB1, PCDHGC3, BTN3A3, BCAP31, CD74, NRP1, ERBB2, EPHB2, EPHB4, EPHA2, PLXND1, TGFBR2, NRP2, SLC3A2-2, ITGA6, CD55- isoform 5, HK2, NECTIN2-isoform 2, R0B01, SLC12A4-isoform 7, TENM1, DYSF, ATP11C, SEMA6B, OCLN, ICAM2, NRG1, SNAP23, RELL1, SDC1, SLC16A7, EMP3, NPTN, or a combination thereof. In some aspects, the labeled protein(s) or a proximity signature comprises, consists of, or consists essentially of VCAM-1, SLC3A2-1, ICAM2, and SLC3 A2-2. In some aspects, the labeled protein(s) or a proximity signature comprises, consists of, or consists essentially of EPHB2, EPHB4, EPHA2, EPHB3, EPHA4, ERBB2, R0B01, PLXND1, TGFBR2, NRP1, NRP2, and SLIT2. In some aspects, the labeled protein(s) or a proximity signature comprises, consists of, or consists essentially of EPHB3, EPHA4, R0B01, and SLIT2. In some aspects, the labeled protein(s) or a proximity signature comprises, consists of, or consists essentially of FAP, CD44, TGFB1, and CTNNB1.

[0016] In some aspects, methods of the disclosure relate, at least in part, to identifying one or more therapeutic targets in a patient. In some aspects, the therapeutic target(s) are proximal to the protein of interest in the sample. In some aspects, the protein of interest is an integrin and / or an integrin complex. In some aspects, the therapeutic target(s) are within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447,448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 angstroms, or any range derivable therein, of the protein of interest in the sample. In some aspects, the therapeutic target(s) are within approximately 200 angstroms of the protein of interest in the sample.

[0017] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0018] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0019] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” “(x and z) or y,” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspect.

[0020] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as“contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0021] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that aspects and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0022] As used herein the term "retrievable moiety" refers to a moiety that can specifically bind to its molecular binding partner to isolate or purify the retrievable moiety and compositions to which it is bound, from a complex mixture. The binding can be through covalent or non-covalent (e.g., ionic, hydrogen, etc.) bonds.

[0023] The term "peptide," “polypeptide,” or “protein” as used herein refers to a polymer of amino acid residues, wherein the polymer can optionally further contain a moiety or moi eties that do not consist of amino acid residues (e.g., an alkyl group, an aralkyl group, an aryl group, a protecting group, or a synthetic polymer, such as, but not limited to a biocompatible polymer). The term applies to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0024] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

[0025] The term "amino acid residue" as used herein refers to a monovalent amino acid or derivative thereof. In some aspects, the term "amino acid residue" refers to the group - NHC(R')C(=O)OR", wherein R' is an amino acid side chain or protected derivative thereof and wherein R" is H or a carboxylic acid protecting group, e.g., methyl.

[0026] The terms "high throughput protein identification," "proteomics" and other related terms are used herein to refer to the processes of identification of a large number or (in some cases, all) proteins in a certain protein sample. Post-translational protein modifications and quantitative information can also be assessed by such methods. One example of "high throughput protein identification" is a gel-based process that includes the pre-fractionation and purification of proteins by one-dimensional protein gel electrophoresis. The gel can then be fractionated into several molecular weight fractions to reduce sample complexity, and proteins can be in-gel digested with trypsin. The tryptic peptides are extracted from the gel, further fractionated by liquid chromatography and analyzed by mass spectrometry. In another approach, a sample can be fractionated without using the gels. For example, by protein extraction followed by liquid chromatography. The proteins can then be digested in-solution, and the proteolytic fragments further fractionated by liquid chromatography and analyzed by mass spectrometry.

[0027] The expression "stable isotope labeling by amino acids in cell culture" (SILAC) is used herein to refer to an approach for incorporation of a label into proteins for mass spectrometry (MS)- based quantitative proteomics. SILAC comprises metabolic incorporation of a given "light" or "heavy" form of the amino acid into the proteins. For example, SILAC comprises the incorporation of amino acids with substituted stable isotopic nuclei (e.g. deuterium,13C,15N) In an illustrative SILAC experiment, two cell populations are grown in culture media that are identical, except that one of them contains a "light" and the other a "heavy" form of a particular amino acid (for example,12C and13C labeled L-lysine, respectively). When the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After a number of cell divisions, each instance of the amino acid is replaced by its isotope-labeled analog. Since there is little chemical difference between the labeled amino acid and the natural amino acid isotopes, the cells behave substantially similar to the control cell population grown in the presence of a normal amino acid.

[0028] As used herein, the term "mass spectrometry" (MS) refers to a technique for the identification and / or quantitation of molecules in a sample. MS includes ionizing the molecules in a sample, forming charged molecules; separating the charged molecules according to their mass-to-charge ratio; and detecting the charged molecules. MS allows for both the qualitative and quantitative detection of molecules in a sample. The molecules can be ionized and detected by any suitable means known to one of skill in the art. Some examples of mass spectrometry are "tandem mass spectrometry" or "MS / MS," which are the techniques wherein multiplerounds of mass spectrometry occur, either simultaneously using more than one mass analyzer or sequentially using a single mass analyzer. The term "mass spectrometry" can refer to the application of mass spectrometry to protein analysis. In some aspects, electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI) can be used in this context. In some aspects, intact protein molecules can be ionized by the above techniques, and then introduced to a mass analyzer. Alternatively, protein molecules can be broken down into smaller peptides, for example, by enzymatic digestion by a protease, such as trypsin. Subsequently, the peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry.

[0029] As used herein, the term "mass spectrometer" is used to refer an apparatus for performing mass spectrometry that includes a component for ionizing molecules and detecting charged molecules. Various types of mass spectrometers can be employed in the methods of the presently disclosed subject matter. For example, whole protein mass spectroscopy analysis can be conducted using time-of-flight (TOF) or Fourier transform ion cyclotron resonance (FT- ICR) instruments. For peptide mass analysis, MALDI time-of-flight instruments can be employed, as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace. Multiple stage quadrupole-time-of-fhght and the quadrupole ion trap instruments can also be used.

[0030] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0031] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0032] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and aspects are discussed throughout this application. Any aspect or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0033] Certain embodiments of the present disclosure are characterized through the following enumerated aspects.

[0034] Aspect l is a photo-dependent proximity labeling system comprising: an activatable probe comprising a ligand covalently attached to a photosensitizer molecule; and a retrievable substrate, wherein the activatable probe and the retrievable substrate are different molecules.

[0035] Aspect 2 is the photo-dependent proximity labeling system of aspect 1, wherein the ligand comprises an Arg-Gly-Asp peptide, or a functional derivative thereof.

[0036] Aspect 3 is the photo-dependent proximity labeling system of aspect 1, wherein the ligand consists of an Arg-Gly-Asp peptide, or a functional derivative thereof.

[0037] Aspect 4 is the photo-dependent proximity labeling system of aspect 1, wherein the ligand comprises a peptide, small molecule, or biologic.

[0038] Aspect 5 is the photo-dependent proximity labeling system of any one of aspects 1-4, wherein the photosensitizer molecule comprises lumichrome, or a functional derivative thereof.

[0039] Aspect 6 is the photo-dependent proximity labeling system of any one of aspects 1-5, wherein the retrievable substrate comprises a structure of BP, AP, SI, S2, S3 or S4:

[0040] Aspect 7 is the photo-dependent proximity labeling system of any one of aspects 1-6, wherein the retrievable substrate comprises a biotin linked to a phenol derivative, a diazirine derivative, a benzophenone derivative, an amine derivative, or an aryl azide derivative.

[0041] Aspect 8 is the photo-dependent proximity labeling system of any one of aspects 1-7, wherein the activatable probe comprises a molecule having a structure of Formula (I):Formula I

[0042] Aspect 9 is the photo-dependent proximity labeling system of any one of aspects 1-8, wherein the retrievable substrate comprises biotin phenol.

[0043] Aspect 10 is the photo-dependent proximity labeling system of any one of aspects 1-9, wherein the ligand is an integrin ligand.

[0044] Aspect 11 is the photo-dependent proximity labeling system of any one of aspects 1-10, wherein the photoactivatable probe is activatable by light.

[0045] Aspect 12 is the photo-dependent proximity labeling system of any one of aspects1-11, wherein the photoactivatable probe is activatable by light of a wavelength of 440 and / or365 nm.

[0046] Aspect 13 is a photo-dependent proximity labeling system comprising a retrievable substrate; and a molecule having a structure of Formula (I):Formula I

[0047] Aspect 14 is the photo-dependent proximity labeling system of aspect 13, wherein the retrievable substrate comprises a structure of formula BP:Biotin Phenol (BP)

[0048] Aspect 15 is a method of labeling a protein complex in a sample, the method comprising: introducing the photo-dependent proximity labeling system of any one of aspects 1-13 to the sample; and exposing the sample to a wavelength of light capable of activating the activatable probe.

[0049] Aspect 16 is the method of aspect 15, wherein introducing the photo-dependent proximity labeling system comprises introducing an activatable probe and a retrievable substrate prior to exposing the sample to light.

[0050] Aspect 17 is the method of aspect 15 or 16, wherein introducing the photodependent proximity labeling system comprises the sequential steps of: introducing an activatable probe, introducing a retrievable substrate, and exposing the sample to light.

[0051] Aspect 18 is the method of any one of aspects 15-17, wherein the protein complex comprises an integrin.

[0052] Aspect 19 is the method of aspect 15 or 18, wherein the sample is comprises a population of cells.

[0053] Aspect 20 is the method of aspect 19, wherein the population of cells comprise a population of mammalian cells.

[0054] Aspect 21 is the method of any one of aspects 15-20, wherein the sample is a biological sample from a patient.

[0055] Aspect 22 is the method of any one of aspects 15-21, wherein the biological sample comprises a fresh sample, a frozen sample, and / or a fixed sample.

[0056] Aspect 23 is the method of any one of aspects 15-21, wherein the biological sample is not frozen and / or not fixed.

[0057] Aspect 24 is the method of any one of aspects 15-23, wherein the sample is a biopsy sample.

[0058] Aspect 25 is the method of any one of aspects 15-24, wherein the sample is from an individual having, suspected of having, or diagnosed with having cancer.

[0059] Aspect 26 is the method of any one of aspects 15-25, wherein the biological sample comprises tissue having a size of approximately 300-500 mm3.

[0060] Aspect 27 is the method of any one of aspects 15-26, wherein the photo-dependent proximity labeling system is introduced at a concentration of at least, at most, approximately, or exactly 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441,442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,499, 500 pM, or any range derivable therein.

[0061] Aspect 28 is the method of any one of aspects 15-27, wherein the photo-dependent proximity labeling system comprises an activatable probe introduced at a concentration of approximately 0.5-50 pM.

[0062] Aspect 29 is the method of any one of aspects 15-27, wherein the photo-dependent proximity labeling system comprises an activatable probe introduced at a concentration of approximately 5-10 pM.

[0063] Aspect 30 is the method of any one of aspects 15-28, wherein the photo-dependent proximity labeling system comprises an activatable probe introduced at a concentration of approximately 50 pM.

[0064] Aspect 31 is the method of any one of aspects 15-30, wherein the photo-dependent proximity labeling system comprises a retrievable substrate introduced at a concentration of approximately 250 pM.

[0065] Aspect 32 is the method of any one of aspects 15-31, wherein the wavelength of light is 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487,488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 nm, or any range derivable therein.

[0066] Aspect 33 is the method of any one of aspects 15-32, wherein the wavelength of light is 440 nm.

[0067] Aspect 34 is the method of any one of aspects 15-32, wherein the wavelength of light is 365 nm.

[0068] Aspect 35 is the method of any one of aspects 15-34, wherein the exposing occurs for between approximately 1 second to 5 minutes.

[0069] Aspect 36 is the method of any one of aspects 15-35, further comprising retrieving proteins labeled with the retrievable substrate from the sample.

[0070] Aspect 37 is the method of aspect 36, wherein the retrieving comprises streptavidin enrichment.

[0071] Aspect 38 is the method of any one of aspects 15-37, further comprising analyzing the sample to determine an identity of proteins labeled with the retrievable substrate.

[0072] Aspect 39 is the method of aspect 38, wherein the analyzing comprises performing mass spectrometry, chromatography, electrophoresis, and / or a suitable protein identification assay.

[0073] Aspect 40 is a method of labeling a protein complex in a sample, the method comprising the steps of: introducing a retrievable substrate comprising biotin and a molecule having a structure of Formula (I) to the sample; exposing the sample to a wavelength of light capable of activating the structure of Formula (I) thereby generating a set of biotin labeled proteins; retrieving the set of labeled proteins with a streptavidin enrichment to generate a set of retrieved proteins; and analyzing the set of retrieved proteins, wherein Formula (I) is:Formula I

[0074] Aspect 41 is the method of aspect 40, wherein the retrievable substrate comprises a formula of BP:Biotin Pheno) (BP)

[0075] Aspect 42 is the method of aspect 40 or 41, wherein the retrievable substrate and the molecule having a structure of Formula (I) are introduced prior to exposing the sample to light.

[0076] Aspect 43 is the method of any one of aspects 40-42, wherein the steps are sequential.

[0077] Aspect 44 is the method of any one of aspects 40-43, wherein the wavelength of light is approximately 440 nm.

[0078] Aspect 45 is the method of aspect 40 or 41, wherein the wavelength of light is approximately 365 nm.

[0079] Aspect 46 is the method of any one of aspects 40-45, wherein the retrievable substrate is introduced at a concentration of approximately 250 pM.

[0080] Aspect 47 is the method of any one of aspects 40-46, wherein the molecule having a structure of Formula (I) is introduced at a concentration of approximately 0.5-50 pM.

[0081] Aspect 48 is the method of any one of aspects 40-47, wherein the molecule having a structure of Formula (I) is introduced at a concentration of approximately 5-10 pM.

[0082] Aspect 49 is the method of any one of aspects 40-48, wherein the sample is a biological sample from a patient.

[0083] Aspect 50 is the method of any one of aspects 40-49, wherein the biological sample comprises a fresh sample, a frozen sample, and / or a fixed sample.

[0084] Aspect 51 is the method of any one of aspects 40-50, wherein the biological sample is not frozen and / or not fixed.

[0085] Aspect 52 is the method of any one of aspects 40-51, wherein the sample is a biopsy sample.

[0086] Aspect 53 is the method of any one of aspects 40-52, wherein the sample is from an individual having, suspected of having, having symptoms of, or diagnosed with having cancer.

[0087] Aspect 54 is a method of identifying one or more therapeutic targets in a patient, the method comprising: introducing the photo-dependent proximity labeling system of any one of aspects 1-13 to a biological sample from the patient, wherein the activatable probe binds to a protein of interest; and exposing the sample to a wavelength of light capable of activating the activatable probe to generate a set of labeled proteins.

[0088] Aspect 55 is the method of aspect 54, wherein introducing the photo-dependent proximity labeling system comprises introducing an activatable probe and a retrievable substrate prior to exposing the sample to light.

[0089] Aspect 56 is the method of aspect 54 or 55, wherein introducing the photodependent proximity labeling system comprises the sequential steps of: introducing an activatable probe, introducing a retrievable substrate, and exposing the sample to light.

[0090] Aspect 57 is the method of any one of aspects 54-56, wherein the set of labeled proteins comprises at least one integrin.

[0091] Aspect 58 is the method of aspect 54 or 57, wherein the biological sample is a biopsy sample.

[0092] Aspect 59 is the method of any one of aspects 54-58, wherein the biological sample comprises a fresh sample, a frozen sample, and / or a fixed sample.

[0093] Aspect 60 is the method of any one of aspects 54-58, wherein the biological sample is not frozen and / or not fixed.

[0094] Aspect 61 is the method of any one of aspects 54-60, wherein the biological sample comprises tissue having a size of approximately 300-500 mm3.

[0095] Aspect 62 is the method of any one of aspects 54-61, wherein the photo-dependent proximity labeling system is introduced at a concentration of at least, at most, approximately, or exactly 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 29,30, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109^ 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128:129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147^ 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 pM, or any range derivable therein.

[0096] Aspect 63 is the method of any one of aspects 54-62, wherein the photo-dependent proximity labeling system comprises an activatable probe introduced at a concentration of approximately 0.5-50 pM.

[0097] Aspect 64 is the method of any one of aspects 54-62, wherein the photo-dependent proximity labeling system comprises an activatable probe introduced at a concentration of approximately 5-10 pM.

[0098] Aspect 65 is the method of any one of aspects 54-64, wherein the photo-dependent proximity labeling system comprises a retrievable substrate introduced at a concentration of approximately 250 pM

[0099] Aspect 66 is the method of any one of aspects 54-65, wherein the wavelength of light is 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487,488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 nm, or any range derivable therein.

[0100] Aspect 67 is the method of any one of aspects 54-66, wherein the wavelength of light is approximately 440 nm.

[0101] Aspect 68 the method of any one of aspects 54-66, wherein the wavelength of light is approximately 365 nm.

[0102] Aspect 69 is the method of any one of aspects 54-68, wherein the exposing occurs for between approximately 1 second to 5 minutes.

[0103] Aspect 70 is the method of any one of aspects 54-69, wherein the patient has, is suspected of having, or has been diagnosed with having cancer.

[0104] Aspect 71 the method of aspect 70, wherein the cancer originated in tissue from bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus in the patient.

[0105] Aspect 72 is the method of aspect 70 or 71, wherein the cancer is, or is suspected of being, metastatic.

[0106] Aspect 73 is the method of any one of aspects 54-72, further comprising retrieving proteins labeled with the retrievable substrate from the sample.

[0107] Aspect 74 is the method of aspect 73, wherein the retrieving comprises streptavidin enrichment.

[0108] Aspect 75 is the method of any one of aspects 54-74, further comprising analyzing the sample to determine an identity of proteins labeled with the retrievable substrate.

[0109] Aspect 76 is the method of aspect 75, wherein the analyzing comprises performing mass spectrometry, chromatography, electrophoresis, and / or a suitable protein identification assay.

[0110] Aspect 77 is the method of any one of aspects 54-76, wherein the therapeutic targets are proximal to the protein of interest in the sample.

[0111] Aspect 78 is the method of any one of aspects 54-77, wherein the therapeutic targets are within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461,462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499,500 angstroms of the protein of interest in the sample.

[0112] Aspect 79 is the method of any one of aspects 54-78, wherein the therapeutic targets are within approximately 200 angstroms of the protein of interest in the sample.

[0113] Aspect 80 is the method of any one of aspects 54-79, wherein the protein of interest comprises an integrin.

[0114] Aspect 81 is the method of any one of aspects 54-80, wherein the protein of interest comprises an oncogenic protein.

[0115] Aspect 82 is an engineered activatable probe comprising a ligand covalently attached to a photosensitizer molecule, wherein the ligand comprises a peptide, small molecule, or biologic; and the photosensitizer molecule comprises lumichrome or a functional derivative thereof.

[0116] Aspect 83 is the engineered activatable probe of aspect 82, wherein the activatable probe comprises a molecule having a structure of Formula (I):Formula I

[0117] Aspect 84 is a method of diagnosing cancer in a patient, said method comprising: introducing the photo-dependent proximity labeling system of any one of aspects 1-13 to a biological sample obtained from the patient, wherein the activatable probe binds to a protein of interest; exposing the sample to a wavelength of light capable of activating the activatable probe to generate a set of labeled proteins; and determining the identity of one or more proteins in the set of labeled proteins.

[0118] Aspect 85 is the method of aspect 84, wherein introducing the photo-dependent proximity labeling system comprises introducing an activatable probe and a retrievable substrate prior to exposing the sample to light.

[0119] Aspect 86 is the method of aspect 84 or 85, wherein introducing the photodependent proximity labeling system comprises the sequential steps of: introducing an activatable probe, introducing a retrievable substrate, and exposing the sample to light.

[0120] Aspect 87 is the method of any one of aspects 84-86, wherein the patient is a human patient.

[0121] Aspect 88 is the method of aspect 84 or 87, wherein the biological sample comprises tissue from a cancer.

[0122] Aspect 89 is the method of aspect 88, wherein the cancer originated in tissue from the patient, wherein the tissue is bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterine tissue.

[0123] Aspect 90 is the method of aspect 88 or 89, wherein the cancer is a solid cancer.

[0124] Aspect 91 is the method of any one of aspects 88-90, wherein the cancer is a metastatic cancer.

[0125] Aspect 92 is the method of any one of aspects 84-91, wherein the labeled proteins are enriched at least about 2-fold.

[0126] Aspect 93 is the method of any one of aspects 84-92, wherein the protein of interest is an integrin.

[0127] Aspect 94 is the method of aspect 93, wherein the labeled proteins comprise ITGB4- isoform 2, CD55, MELTF, NECTIN2, DLG1, PTPRK, CSF2RA, FRMD6, VASN, FCGRT, PCDHB13, CA12, HRH1, SLC3A2-1, VCAM-1, EPHB3, EPHA4, SLIT2, FAP, CD44, TGFB1, CTNNB1, PCDHGC3, BTN3A3, BCAP31, CD74, NRP1, ERBB2, EPHB2, EPHB4, EPHA2, PLXND1, TGFBR2, NRP2, SLC3A2-2, ITGA6, CD55-isoform 5, HK2, NECTIN2- isoform 2, ROBO1, SLC12A4-isoform 7, TENM1, DYSF, ATP11C, SEMA6B, OCLN, ICAM2, NRG1, SNAP23, RELL1, SDC1, SLC16A7, EMP3, NPTN, or a combination thereof.

[0128] Aspect 95 is the method of any one of aspects 84-94, further comprising administering an effective amount of a treatment to the patient.

[0129] Aspect 96 is the method of aspect 95, wherein the treatment comprises an inhibitor of ITGB4-isoform 2, CD55, MELTF, NECTIN2, DLG1, PTPRK, CSF2RA, FRMD6, VASN, FCGRT, PCDHB13, CA12, HRH1, SLC3A2-1, VCAM-1, EPHB3, EPHA4, SLIT2, FAP,CD44, TGFB1, CTNNB1, PCDHGC3, BTN3A3, BCAP31, CD74, NRP1, ERBB2, EPHB2, EPHB4, EPHA2, PLXND1, TGFBR2, NRP2, SLC3A2-2, ITGA6, CD55-isoform 5, HK2, NECTIN2-isoform 2, R0B01, SLC12A4-isoform 7, TENM1, DYSF, ATP11C, SEMA6B, OCLN, ICAM2, NRG1, SNAP23, RELL1, SDC1, SLC16A7, EMP3, NPTN, or a combination thereof

[0130] Aspect 97 is the method of aspect 95 or 96, wherein the treatment is an anti- metastatic treatment.

[0131] Aspect 98 is a method of identifying binding partners of a protein of interest in a biological sample obtained from a patient, said method comprising: introducing the photodependent proximity labeling system of any one of aspects 1-13 to the biological sample wherein the activatable probe binds to the protein of interest; exposing the sample to a wavelength of light capable of activating the activatable probe to generate a set of labeled proteins.

[0132] Aspect 99 is the method of aspect 98, wherein introducing the photo-dependent proximity labeling system comprises introducing an activatable probe and a retrievable substrate prior to exposing the sample to light.

[0133] Aspect 100 is the method of aspect 98 or 99, wherein introducing the photodependent proximity labeling system comprises the sequential steps of: introducing an activatable probe, introducing a retrievable substrate, and exposing the sample to light.

[0134] Aspect 101 is the method of any one of aspects 98-100, wherein the set of labeled proteins are identified as proteins that bind to the protein of interest.

[0135] Aspect 102 is the method of aspect 101, wherein the biological sample obtained from a patient is obtained from a human patient.

[0136] Aspect 103 is the method of any one of aspects 98-102, wherein the biological sample obtained from a patient comprises tissue from a cancer.

[0137] Aspect 104 is the method of aspect 103, wherein the cancer originated in tissue from the patient, wherein the tissue is bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterine tissue.

[0138] Aspect 105 is the method of aspect 103 or 104, wherein the cancer is a solid cancer.

[0139] Aspect 106 is the method of any one of aspects 103-105, wherein the cancer is a metastatic cancer.

[0140] Aspect 107 is the method of any one of aspects 98-106, wherein the binding partners are enriched at least about 2-fold.

[0141] Aspect 108 is the method of any one of aspects 98-107, wherein the protein of interest is an integrin.

[0142] Aspect 109 is the method of any one of aspects 98-108, wherein the labeled proteins comprise ITGB4-isoform 2, CD55, MELTF, NECTIN2, DLG1, PTPRK, CSF2RA, FRMD6, VASN, FCGRT, PCDHB13, CA12, HRH1, SLC3A2-1, VCAM-1, EPHB3, EPHA4, SLIT2, FAP, CD44, TGFB1, CTNNB1, PCDHGC3, BTN3A3, BCAP31, CD74, NRP1, ERBB2, EPHB2, EPHB4, EPHA2, PLXND1, TGFBR2, NRP2, SLC3A2-2, ITGA6, CD55-isoform 5, HK2, NECTIN2-isoform 2, ROBO1, SLC12A4-isoform 7, TENM1, DYSF, ATP11C, SEMA6B, OCLN, ICAM2, NRG1, SNAP23, RELL1, SDC1, SLC16A7, EMP3, NPTN, or a combination thereof.

[0143] Aspect 110 is the method of any one of aspects 98-109, wherein the labeled proteins comprise VCAM-1, SLC3A2-1, ICAM2, and SLC3A2-2.

[0144] Aspect I l l is the method of any one of aspects 98-109, wherein the labeled proteins comprise EPHB3, EPHA4, ROBO1, and SLIT2.

[0145] Aspect 112 is the method of any one of aspects 98-109, wherein the labeled proteins comprise FAP, CD44, TGFB1, and CTNNB1.

[0146] Aspect 113 is the method of any one of aspects 98-109, wherein the labeled proteins comprise EPHB2, EPHB4, EPHA2, EPHB3, EPHA4, ERBB2, ROBO1, PLXND1, TGFBR2, NRP1, NRP2, and SLIT2.

[0147] It is specifically contemplated that any limitation discussed with respect to one aspect or aspect of the invention may apply to any other aspect or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an aspect set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of the Invention, Brief Description of the Drawings, Detailed Description of the Invention, and / or Claims.

[0148] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications withinthe spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0149] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0150] FIG. 1 - RGDL-dependent photoproximity profiling (RGDL-PhotoPPI) of native integrin complexes. FIG. 1A shows chemical structures of lumichrome-NHS (1) and RGDL photoproximity probe (left). As shown, lumichrome functionalized chemical probes can be used to photosensitize an inert phenolic substrate into the reactive phenoxyl radical at RGDL-bound complexes in situ (right). FIG. IB shows a schematic of RGDL-PhotoPPI workflow on live cells. FIG. 1C shows detection of biotinylated bovine serum albumin (BSA) by immunoblot (a-biotin) and Coomassie gel in response to RGDL workflow (top), and quantification of biotin phenol (BP) substrate concentration-dependent biotinylation (bottom). FIG. ID shows an immunoblot quantification of time-dependent BSA photolabeling with 440 nm (circles) and 365 nm (squares) light activation. FIG. IE shows anti -biotin (a-biotin) immunoblot of U87-MG cells incubated with different RGDL ligand concentrations, followed by BP substrate treatment and blue light irradiation. FIG. IF shows streptavidin-stained immunoblot (left) of RGDL photoprobed live U87-MG and HEK293T cell cultures, and quantification of total lane intensity for each dose of RGDL tested (right). Data in FIGs. 1C- 1F are representative samples from n = 2 biological replicates. P-values in FIG. IF are from Student’ s two-way t-test.

[0151] FIG. 2 - RGDL-PhotoPPI detected conserved and unique integrin interactomes on live glioblastoma and cervical cancer cells. FIG. 2A shows a schematic depicting probe vs. no-probe RGDL-PhotoPPI target identification using SILAC-labeled cells. FIG. 2B shows a waterfall plot of SILAC ratios (RGDL-treated / DMSO-treated) from U87- MG cells as described in FIG. 2A. Raw enrichment of > 4-fold (i.e., RGDL probe-dependent labeling) in two or more biological replicates (from n = 4 total) was used as a cutoff for identifying proximal targets. Representative integrin (“Integrins”), growth factor-related (“Growth Factor and Receptors”), focal adhesion member (“Focal Adhesions”), and surface protease (“Proteases”) proteins are highlighted. FIG. 2C shows a Yang plot depicting therelationship between the level of enrichment (median ion intensity) and the number of STRING database-annotated protein-protein interactions (PPIs) for each protein enriched from analysis in FIG. 2B. Highly enriched proteins with the most abundant connectivity appear in the top right of the plot. Integrins (marked in the plot) and proteins that are one standard deviation (SD) above the mean logio (‘Heavy’ grouped ion intensity; Z-score >1) are highlighted. FIG. 2D shows U87-MG (top) and HeLa (bottom) connectivity plots of enriched proteins from the ‘integrin binding’ gene ontology molecular function (GOMF) group. FIG. 2E shows a heatmap depiction of unique and common integrin interaction partners identified in FIGs. 2B-2C from U87-MG and HeLa (left), and memberships within select pathways (right). A subset of enriched proteins is shown on the right. FIG. 2F shows a heatmap of unique and conserved enriched GO, KEGG, and HALLMARK pathway terms from Metascape analysis of the data in FIG. 2E. FIG. 2G shows a graphic depiction of some conserved aP-integrin social network members identified by RGDL-PhotoPPI profiling in U87-MG and HeLa cancer cell lines.

[0152] FIG. 3 - RGDL-PhotoPPI mapping in diverse cellular contexts. FIG. 3 A shows a schematic of attached and detached conditions used for RGDL-PhotoPPI profiling. FIG. 3B shows a principal component analysis (PCA) of integrin interacting members in adherent and detached cultures of HeLa and U87-MG cells. FIG. 3C shows heatmaps showing a subset of integrin-binding associated protein ratios that were consistent and unique to adherent (“Adh .”) and detached (“Det.”) cultures. FIGs. 3D-3E show a GOMF ‘integrin binding’ STRING network (FIG. 3D) and a Yang Plot (FIG. 3E) of RGDL-enriched proteins from detached U87- MG cells. FIG. 3F shows radar plots comparing gene set enrichment P-values of attached and detached U87-MG cells. GO Molecular Function terms (left) and GO Cellular Component terms (right). Data shown in FIGs. 3B-3F represent n = 3 biological replicates for detached U87-MG and HeLa cell cultures. FIG. 3G shows a schematic of RGDL-PhotoPPI tissue profiling of mouse tumor xenografts (left) and anti-biotin immunoblot analysis of MDA-MB- 231 xenografts treated with RGDL probe and processed as indicated from n = 2 biological replicates (right). FIGs. 3H-3I show a Yang Plot (FIG. 3H) of RGDL-PhotoPPI-enriched proteins from U87-MG tumor xenografts and a STRING network (FIG. 31) of proteins in the ‘integrin binding’ gene ontology molecular function (GOMF) category.

[0153] FIG. 4 - Unique integrin interaction networks typified TNBC cell line metastatic stage. FIG. 4A shows a schematic depiction of a xenograft model using MDA- MB-231 (“231”) from which BM1 cells can be obtained after metastasis (top) and a schematic of SILAC -based RGDL-PhotoPPI profiling of surface interactors therein. FIG. 4B shows a volcano plot comparing relative abundance (via RGDL-PhotoPPI SILAC ratio) of integrinsand interaction partners on live MDA-MB-231 and BM1 cells; n = 4 biological replicates. FIGs. 4C-4E shows quantitative Proximity Scores in 231 (left bar) and BM1 (right bar) cells for previously known integrin interacting proteins (FIG. 4C) and novel integrin interactors in the Ephrin (FIG. 4D) and NRP / ROBO (FIG. 4E) receptor families. FIG. 4F shows a STRING network depiction of angiogenesis-related integrin interactors detected in 231 and BM1 cells. FIG. 4G shows a schematic depiction of differential Ephrin- and NRP / ROBO sub-network interactions with aP-integrin complexes in MDA-MB-231 (“231”) and BM1 cells. Data in FIGs. 4B-4E represent mean ± S.E.M. from n = 4 biological replicates. Statistical comparisons are two-tailed unpaired t-tests.

[0154] FIG. 5 - Expression of primary tumor- or metastatic tumor-specific integrin interactors correlated with patient outcomes in human breast cancers. FIG. 5A shows a Kaplan-Meier (KM) analysis of triple-negative breast cancer (TNBC) patient survival based on relative mRNA expression levels of unique 20-gene signatures derived from MDA-MB-231 cells (left) or BM1 cells (right). FIG. 5B KM analysis of patients from all breast cancers in the KM database. Survival data of patients with higher aggregate barcode expression are labeled “ii”, and those grouped by lower expression are labeled “i”. Hazard ratios (HR) with 95% confidence intervals and logrank P-values are indicated below each survival plot. FIG. 5C shows a schematic of RGDL-PhotoPPI for the in situ ligand directed proximity labeling of native integrins to identify disease relevant protein interactome barcodes between cell types.

[0155] FIG. 6 - Lumichrome photosensitization of biotin-tagged substrates. FIG. 6A shows quantification of an immunoblot demonstrating dose dependence of photocatalyst 2 (PC 2) on BP photosensitization. FIG. 6B shows an immunoblot (top) and quantification (bottom) of BSA photolabeling with PC2 and BP at 365 nm. FIG. 6C shows chemical structures of substrate probes: SI, S2, S3 and S4. FIG. 6D shows a screen of substrates S1-S4 photosensitization with PC 2; immunoblot (top) and quantification (bottom). FIG. 6E shows a dose response of SI (top) and S4 (bottom) in the presence of vehicle or PC 2, and 440 nm light.

[0156] FIG. 7 - STRING analysis of HeLa cells in attached and detached cellular states. FIG. 7A shows a Yang plot of RGDL-enriched proteins (left) and GOMF ‘integrin binding’ STRING network of enriched proteins from attached HeLa cells (right); n = 4. FIG. 7B shows a Yang plot of RGDL-enriched proteins (left) and GOMF ‘integrin binding’ STRING network of enriched proteins from detached HeLa cells (right); n = 3. FIG. 3C shows radial plots of GO Molecular function (left) and GO Cellular Component (right) terms from RGDL- PhotoPPI profiling of adhered and detached HeLa cells.

[0157] FIG. 8 - Rapid integrin interactome profiling of breast cancer-derived tumor xenografts. FIG. 8A shows a schematic of RGDL-PhotoPPI tissue profiling workflow. FIG. 8B shows a Yang plot of RGDL-enriched proteins from a MDA-MB-231 tumor xenograft; n = 1. FIG. 8C shows a GOMF ‘integrin binding’ STRING network of enriched proteins from MDA-MB-23 1 tumor xenograft.

[0158] FIG. 9 - STRING analysis of RGDL-PhotoPPI profiling of MDA-MB-231 and BM1 cells. FIG. 9A shows a Yang plot of RGDL-enriched proteins (left) and GOMF ‘integrin binding’ STRING network of enriched proteins from MDA-MB-231 cells (right); n = 4. FIG. 9B shows a Yang plot of RGDL-enriched proteins (left) and GOMF ‘integrin binding’ STRING network of enriched proteins from BM1 cells (right); n = 4. FIG. 9C shows a heatmap of unique GO, KEGG, and HALLMARK pathway terms from Metascape analysis of the data in FIGs. 9A-9B. FIG. 9D shows bar plots of SILAC ratio, median RGDL ion intensity, and proximity scores for ITGB3, EGFR and VCAM1. Data represents mean ± S.E.M. from n = 4 biological replicates. FIG. 9E shows 20 gene survival signature lists for MDA-MB-231 (left) and BM1 (right).

[0159] FIG. 10 - Whole proteome analysis of MDA-MB-231 and BM1 cells. FIG. 10A shows a volcano plot of proteome abundance comparison of MDA-MB-231 and BM1 cells from n = 4 biological replicates. Integrins are labeled “i” and ‘integrin binding’ (GOMF) proteins are labeled “ii”. Cutoffs for ratio and p-value are represented by a dotted line indicating 2-fold change and p-value of 0.05. FIG. 10B shows a plot of ratios for integrins detected and quantified in FIG. 10A.

[0160] FIG. 11 shows a chemical synthesis pathway of an RGDL photoproximity probe of the disclosure.

[0161] FIGs. 12A-12B show LC-MS spectra of an RGDL of the disclosure.

[0162] FIG. 13 shows a chemical synthesis pathway of norbiotinamine.

[0163] FIG. 14 shows a chemical synthesis pathway of substrate SI (SI).

[0164] FIG. 15 shows a chemical synthesis pathway of substrate S2 (S2).

[0165] FIG. 16 shows a chemical synthesis pathway of substrate S3 (S3).

[0166] FIG. 17 shows a chemical synthesis pathway of substrate S4 (S4).

[0167] FIG. 18 shows an NMR spectra of dinitrobenzoic acid.

[0168] FIG. 19 shows an NMR spectra of lumichrome.

[0169] FIG. 20 shows an NMR spectra of lumichrome-NHS.

[0170] FIG. 21 shows an NMR spectra of S 1.

[0171] FIG. 22 shows an NMR spectra of S2.

[0172] FIG. 23 shows an NMR spectra of S2.

[0173] FIG. 24 shows an NMR spectra of S3.

[0174] FIG. 25 shows an NMR spectra of S4.

[0175] FIG. 26 shows uncropped gels for the data shown in FIG. 6A. FIG. 26 shows aStreptavidin-800 immunoblot (left) and Coomassie (right) from a lumichrome photocatalysis 2 dosing assay (0-20 pM) with 100 pM BP and 5 min of 440 nm light.

[0176] FIGs. 27A-27B show uncropped gels for the data shown in FIG. 1C. FIG. 27 A shows an immunoblot (left) and Coomassie (right) of BP doses (0-500 pM) with 20 pM photocatalyst 2 and 15 min of 440 nm light. FIG. 27B shows an immunoblot (left) and Coomassie (right) of BP doses (0-500 pM) with 20 pM photocatalyst 2 and 15 min of 365 nm light.

[0177] FIGs. 28 shows uncropped gels for the data shown in FIG. ID. FIG. 28 shows a Streptavidin-800 immunoblot (left) and Coomassie (right) of 440 nm light doses (0-900 s) with 20 pM photocatalyst 2 and 100 pM BP.

[0178] FIGs. 29 shows uncropped gels for the data shown in FIG. ID. FIG. 29 shows a Streptavidin-800 immunoblot (left) and Coomassie (right) of 365 nm light doses (0-900 s) with 20 pM photocatalyst 2 and 100 pM BP.

[0179] FIGs. 30 shows uncropped gels for the data shown in FIG. 6D. FIG. 30 shows an immunoblot (left) and Coomassie (right) of SI doses (0-500 pM) with 20 pM photocatalyst 2 and 5 min of 440 nm light.

[0180] FIGs. 31 shows an uncropped gel for the data shown in FIG. 6D. FIG. 31 shows an immunoblot of S4 doses (0-500 pM) with 20 pM photocatalyst 2 and 5 min of 440 nm light.

[0181] FIGs. 32 shows an uncropped gel for the data shown in FIG. 6C. FIG. 32 shows an immunoblot of a photolabeling assay comparing BP, SI, S2, S3, and S4 at 100 pM in the presence of 20 pM photocatalyst 2 and 5 min of 440 nm light.

[0182] FIGs. 33 shows an uncropped gel for the data shown in FIG. IE. FIG. 33 shows a Streptavidin-800 immunoblot (top) and a Coomassie stain (right) of RGDL probe dosing (0-25 pM) on live U87-MG cells. Cells were treated with 250 pM BP and 440 nm light (1 minute).

[0183] FIGs. 34 shows uncropped gels for the data shown in FIG. 4B. FIG. 34 shows Streptavidin-800 immunoblots for low input (left) and high input (right) of RGDL profiling of MDA 231 tumor tissue.

[0184] FIG. 35 shows an uncropped gel for data shown in FIG. IF. FIG 35 shows a Streptavidin-800 immunoblot for RGDL probe dosing (5 to 10 pM) on live U87-MG (lanes 4-10) and HEK293T (lanes 1-3) cells, which were treated with 250 pM BP and 440 nm light (1 minute).

[0185] FIG. 36 shows chemical structures of probes and substrates of the disclosure.DETAILED DESCRIPTION OF THE INVENTIONI. Photoproximity Probes and Systems

[0186] In some aspects, the presently disclosed subject matter provides a photoactive chemical probe or probe system for use in photoproximity profiling of biological interactions, e.g., protein-protein interactions, protein-metabolite interactions, protein-drug interactions, cell-cell interactions, nucleic acid-drug interactions, etc. The probe or probe system can be used to profile such interactions in or on live cells, thereby eliminating biases against low concentration biological entities, weak interaction affinities, and false-positive interactions caused when studying biological interactions in lysed cells.

[0187] Generally, the presently disclosed photoactive probes or probe systems include, as parts of a single probe molecule or as parts of more than one different probe molecules (e.g., two different probe molecules) designed to be used in combination, a ligand capable of specifically binding (covalently or non-covalently) a first binding partner, a photosensitizer molecule, and a retrievable substrate. The ligand and photosensitizer molecule may be operably linked.

[0188] The ligand can be a moiety that specifically binds a moiety associated with a biological target of interest, such as a protein of interest. For example, the ligand can be a peptide or protein capable of binding a target biological molecule, such as another protein. In some aspects, a ligand binds an integrin. In some aspects, an integrin ligand exhibits a high binding affinity and specificity for av or a5 integrins, such as for the integrins avP3 and / or a5pi. When an integrin ligand is part of a molecule, it confers its property to the molecule, and the said molecule become “targeted” to integrins, i.e. the molecule specifically and efficiently binds to integrins. The binding between integrins and an integrin ligand may be covalent or non-covalent, including, in this last case, hydrophobic interactions, electrostatic interactions, Van der Waals interactions, hydrogen bonding etc. Most often the binding is non-covalent.

[0189] In some aspects, the retrievable substrate comprises a reactive substrate activatable by a photo-activatable probe and a retrievable moiety operably coupled to each other. A retrievable moiety may be a member of a specific binding pair (e.g., biotimavidin, antibody:antigen). In some aspects, a retrievable moiety, such as biotin, can selectively bind toan affinity matrix, such as streptavidin coated beads or particles. The retrievable moiety may also be a peptide tag. Peptide tags include, but are not limited to, chitin binding protein-tag, maltose binding protein-tag, glutathione-S-transferase-tag, polyhistidine (His-tag), FLAG-tag, V5 tag, VSV-tag, Myc-tag, c-Myc-tag, HA-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, Strep-tag, TC tag, calmodulin-tag, Avi-tag, Xpress tag, isopeptag, Spy-tag, biotin carboxyl carrier protein (BCCP), green fluorescent protein-tag, HaloT-tag, Nustag, Fc-tag, Ty tag, thioredoxin-tag, or poly(NANP). In some aspects, the retrievable moiety is biotin or desthiobiotin. In some aspects, the retrievable moiety is selected from the group consisting of: biotin or an analogue thereof; digoxigenin; fluorescein; dinitrophenol; and an immunotag. The reactive substrate can be any suitable substrate that forms a reactive species capable of bonding (covalently or non-covalently) to a protein, peptide, small molecule, or nucleic acid when activated by a photo-activatable probe that has been exposed to light. In some aspects, a photoreactive probe activates a reactive substrate after exposure to a light of approximate wavelength of 440 nm. In some aspects, a photoreactive probe activates a reactive substrate after exposure to a light of approximate wavelength of 365 nm. The reactive substrate may comprises a diazirine derivative, a phenol derivative, a benzophenone derivative, an amine derivative, or an aryl azide derivative.II. Sample Preparation

[0190] In certain aspects, methods involve obtaining a sample from a subject. The methods of obtaining provided herein may include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In certain aspects the sample is obtained from a biopsy from esophageal tissue by any of the biopsy methods previously mentioned. In other aspects the sample may be obtained from any of the tissues provided herein that include but are not limited to non-cancerous or cancerous tissue and non-cancerous or cancerous tissue from the serum, gall bladder, mucosal, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample may be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Yet further, the biological sample can be obtained without the assistance of a medical professional.

[0191] A sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. The biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.

[0192] The sample may be obtained by methods known in the art. In certain aspects the samples are obtained by biopsy. In other aspects the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other methods known in the art. In some cases, the sample may be obtained, stored, or transported using components of a kit of the present methods. In some cases, multiple samples, such as multiple esophageal samples may be obtained for diagnosis by the methods described herein. In other cases, multiple samples, such as one or more samples from one tissue type (for example esophagus) and one or more samples from another specimen (for example serum) may be obtained for diagnosis by the methods. In some cases, multiple samples such as one or more samples from one tissue type (e.g. esophagus) and one or more samples from another specimen (e.g. serum) may be obtained at the same or different times. Samples may be obtained at different times are stored and / or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods.

[0193] In some aspects the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist. The medical professional may indicate the appropriate test or assay to perform on the sample. In certain aspects a molecular profiling business may consult on which assays or tests are most appropriately indicated. In further aspects of the current methods, the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0194] In other cases, the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, endoscopy, or phlebotomy. The method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some aspects, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0195] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is herein incorporated by reference in its entirety, describes general methods for biopsy and cytological methods. In one aspect, the sample is a fine needle aspirate of a esophageal or a suspected esophageal tumor or neoplasm. In some cases, the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.

[0196] In some aspects of the present methods, the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business. In some cases, the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.

[0197] In some aspects of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit. An OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit. In some cases, molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately. A sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and / or adequacy are provided.

[0198] In some aspects, the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist. The specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample. In some cases the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling business may obtain the sample.A. Protein Identification

[0199] In some aspects, a protein of interest is measured. Polypeptides from the protein of interest can be quantified by any methods known to those of skill in the art from samples as described herein. In some aspects, antibodies can also be used to detect polypeptides / proteins of interest. Antibodies to a protein of interest can be produced using well known techniques (see, e.g., Harlow & Lane, 1988 and Harlow & Lane, 1999; Coligan, 1991; Goding, 1986; and Kohler & Milstein, 1975). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., 1989; Ward et al., 1989).

[0200] Once specific antibodies are available, the expression of a protein of interest can be detected by a variety of immunoassay methods. For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (1991). Moreover, the immunoassays of certain aspects can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (1980); and Harlow & Lane, supra).

[0201] Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen. The labeling agent may itself be one of the moieties comprising the antibody / antigen complex. Thus, the labeling agent may be a labeled polypeptide or a labeled antibody that binds the protein of interest. Alternatively, the labeling agent may be a third moiety, such as a secondary antibody, that specifically binds to the antibody / antigen complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived). Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the labeling agent. These proteins exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al., 1973; Akerstrom et al., 1985). The labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art.

[0202] Commonly used assays include noncompetitive assays, e.g., sandwich assays, and competitive assays. In competitive assays, the amount of polypeptide present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) polypeptide of interest displaced (competed away) from an antibody that binds by the unknown polypeptide present in a sample. Commonly used assay formats include immunoblots, which are used to detect and quantify the presence of protein in a sample. Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g.,antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al., 1986).

[0203] Any suitable method can be used to detect one or more of the proteins described herein. Successful practice can be achieved with one or a combination of methods that can detect and, preferably, quantify the proteins. These methods include, without limitation, gel electrophoresis, mass spectrometry (e.g., laser desorption / ionization mass spectrometry), fluorescence (e.g., sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy. Expression levels of proteins of interest can be compared by procedures well known in the art, such as Western blotting, immunoblotting, flow cytometry, immunocytochemistry, binding to magnetic and / or antibody-coated beads, ELISA, multiplex mass spectrometry (e.g., tandem mass tag mass spectrometry), or colorimetric assays.

[0204] Methods may further include, one or more of electrospray ionization mass spectrometry (ESLMS), ESLMS / MS, ESI- MS / (MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCL MS), APCI-MS / MS, APCI-(MS)11, atmospheric pressure photoionization mass spectrometry (APPLMS), APPLMS / MS, and APPI-(MS)n, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero.

[0205] In various aspects, targeted quantification of peptides can incorporate use of liquid chromatography -mass spectrometry LC / MS instrumentation. For example, LC-MS / MS, or tandem MS may be used. In general, LC / MS e.g., LC-MS / MS) can combine the physical separation capabilities of liquid chromatograph (LC) with the mass analysis capabilities of mass spectrometry (MS). According to some aspects described herein, this technique allows for the separation of digested peptides to be fed from the LC column into the MS ion source through an interface.In various aspects, an instrument or instrument system suited for identification and targeted quantification may include, for example, a Triple Quadrupole LC / MS™. In various aspects, an instrument or instrument system suited for LC may include, for example, an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific), an Acclaim PepMap RSLC C18 column (75 pm x 15 cm, 2 pm, 100 A, Thermo Fisher Scientific), an in-line Acclaim PepMap 100 C18 trap column (75 pm x 2 cm, 3 pm, 100 A, Thermo Fisher Scientific), equivalents thereof, or acombination thereof. In some aspects, an MS system may include, for example, an Orbitrap Exploris 480 and Nanospray Flex Ion Source with stainless steel emitter tip (Thermo Fisher Scientific).

[0206] In some cases, targeted quantification includes using a specific collision energy associated for the appropriate fragmentation to consistently see an abundant product ion.

[0207] In various aspects, quality control procedures can be put in place to optimize data quality. In various aspects, measures can be put in place allowing only errors within acceptable ranges outside of an expected value. In various aspects, employing statistical models (e.g., using Westgard rules) can assist in quality control. For example, quality control may include, for example, assessing the retention time and abundance of representative peptide structures and spiked-in internal standards, in either every sample, or in each quality control sample.

[0208] Peak integration and normalization may be performed to process the data that has been generated and transform the data into a format for analysis. For example, peak integration and normalization may include converting abundance data for various product ions that were detected for a selected peptide structure into a single quantification metric (e.g., a relative quantity, an adjusted quantity, a normalized quantity, a relative concentration, an adjusted concentration, a normalized concentration, etc.) for that peptide structure. In some aspects, peak integration and normalization may be performed using one or more of the techniques described in U.S. Patent Publication No. 2020 / 0372973A1 and / or US Patent Publication No. 2020 / 0240996A1, the disclosures of which are incorporated by reference herein in their entireties.B. Integrins

[0209] In some aspects, a protein of interest is an integrin. Integrins constitute a superfamily of membrane receptors that are encoded by genes belonging to a gene superfamily and are typically composed of heterodynamic transmembrane glycoproteins containing an a- and a P-subunit. The integrin family of cell surface proteins is comprised of 18 distinct a- integrins and eight P-integrins, which form a network of 24 known functional heterodimers. Members of an integrin subfamily have a common P subunit, which can combine with different a subunits to form adhesion protein receptors with different specificities. Among the known a subunits, the av subunit seems to be the most promiscuous, forming heterodimers with six different P subunits. Integrin av include, for example, the receptors avP3 and avP5.III. Administration of Therapeutic Compositions

[0210] The therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapies may be administered in any suitable manner known in the art. For example, the first and second cancer treatment may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, the first and second cancer treatments are administered in a separate composition. In some aspects, the first and second cancer treatments are in the same composition.

[0211] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0212] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some aspects, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0213] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0214] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0215] It will be understood by those skilled in the art and made aware that dosage units of pg / kg or mg / kg of body weight can be converted and expressed in comparable concentrationunits of pg / ml or mM (blood levels). It is also understood that uptake is species and organ / tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0216] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more administrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, or 12 week intervals, including all ranges there between.

[0217] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0218] The active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Typically, such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0219] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0220] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0221] A pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0222] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0223] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0224] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above..A. Cancer Therapy

[0225] In some aspects, the method further comprises administering a cancer therapy to the patient. The cancer therapy may be chosen based on the expression level measurements, alone or in combination with the clinical risk score calculated for the patient. In some aspects, the cancer therapy comprises a local cancer therapy. In some aspects, the cancer therapy excludes a systemic cancer therapy. In some aspects, the cancer therapy excludes a local therapy. In some aspects, the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy. In some aspects, the cancer therapy comprises an immunotherapy, which may be an immune checkpoint therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.

[0226] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain aspects, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some aspects, the cancer is recurrent cancer. In some aspects, the cancer is Stage I cancer. In some aspects, the cancer is Stage II cancer. In some aspects, the cancer is Stage III cancer. In some aspects, the cancer is Stage IV cancer.

[0227] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serouscystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.IV. Kits

[0228] Certain aspects of the present disclosure also concern kits containing compositions of the disclosure or compositions to implement methods disclosed herein. In some aspects, kits can be used to evaluate one or more biomarkers. In some aspects, there are kits for evaluating biomarker activity in a cell.

[0229] Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0230] Individual components may also be provided in a kit in concentrated amounts; in some aspects, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.

[0231] In certain aspects, negative and / or positive control probes are included in some kit aspects. In addition, a kit may include a sample that is a negative or positive control for methylation of one or more biomarkers.

[0232] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different aspects may be combined. The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims.

[0233] Any aspect of the disclosure involving specific biomarker by name is contemplated also to cover aspects involving biomarkers whose sequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified nucleic acid.

[0234] Aspects of the disclosure include kits for analysis of a pathological sample by assessing biomarker profile for a sample comprising, in suitable container means, two or more biomarker probes, wherein the biomarker probes detect one or more of the biomarkers identified herein. The kit can further comprise reagents for labeling nucleic acids in the sample. The kit may also include labeling reagents, including at least one of amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents can include an aminereactive dye.EXAMPLES

[0235] The following examples are included to demonstrate preferred aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed inthe examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.I. Example 1 - Methods

[0236] General synthetic methods: Reagents purchased from commercial suppliers were analytical grade and used without further purification. All reactions were carried out in oven- dried flasks, using oven-dried magnetic stir rods and anhydrous solvents (ACROS®) unless otherwise specified. Nuclear magnetic resonance spectra were acquired using either a BRUKER® AVANCE 11+ 500; 11.7 Tesla NMR or BRUKER® DRX 400; 9.3 Tesla NMR instrument. Accurate mass measurements and final probe purification were obtained and performed using an AGILENT® 1290 Infinity II with an AGILENT® 5 Prep C18 50 x 21.2 mm column.

[0237] Cell culture: The U87-MG cell line was obtained from the University of Chicago Cellular Screening Center. MDA-MB-231 and BM1 cells were gifted by Marsha Rosner (University of Chicago). The MDA-MB-231 cell line was originally obtained from ATCC and the BM1 cell line was generated by Kang and colleagues (Kang et al., 2003) and obtained from Joan Massague (Memorial Sloan-Kettering Cancer Center).

[0238] U87-MG and HeLa cells were propagated in RPMI (CORNING®) supplemented with 10% fetal bovine serum (FBS, CORNING ®) and 1% penicillin / streptomycin (GIBCO®). MDA-MB-231 and BM1 lines were propagated in DMEM (INVITROGEN®). All cell lines were grown at 37 °C in a 5% CO2 humidified incubator. All cell lines tested negative for mycoplasma using a detection kit (MycoAlert PLUS Detection Kit; LONZA®).

[0239] SDS-PAGE and Immunoblot: Cells were lysed in the well using ice-cold 1 x RIP A (MILLIPORE®) and tip sonicated (FB-505, FISHER SCIENTIFIC®) over ice. Insoluble debris was cleared by centrifugation, and the supernatant was diluted using 4X Laemmli buffer containing 50 mM dithiothreitol (DTT) as a reducing agent. Samples were prepared for SDS- PAGE by heating to 95 °C for 5 min, cooled to room temperature, resolved on a 10% SDS- PAGE gel, and transferred to nitrocellulose membranes by standard western blotting methods. Membranes were blocked using 2% BSA in TBS containing 0.1% of a non-ionic detergent (TWEEN®-20) (TBST) and probed with streptavi din-800 (Odyssey CLx, LI-COR®) tovisualize biotin labeling. Blot intensities were quantified in Image J and normalized in MICROSOFT® Excel.

[0240] In vitro photosensitization experiments: A solution of BSA (10 pM) in phosphate- buffered saline (PBS) was prepared for all in vitro photolabeling assays. The optimal concentration of photocatalyst for in vitro photolabeling experiments was determined by performing a dose gradient of photocatalyst 2 (0-20 pM) on solutions of BSA with 100 pM BP with irradiation at 440 nm LED for 5 min. For substrate dosing assays, samples were treated with vehicle or lumichrome photocatalyst 2 (20 pM) from a 5 mM stock solution. BP (0, 50,100, 200, 350, 500 pM) was then added and samples were then irradiated at 440 nm with an LED (PR160L, KESSIL®) or at 365 nm with a UV crosslinker (XL-1500, SPECTROLINE®) for 5 min on ice. For photokinetic experiments, BSA solutions with 100 pM BP and 20 pM photocatalyst 2 were irradiated with either wavelength for 0, 30, 60, 120, 180, 240, 300, 600, or 900 seconds (s), with samples collected at each time point. Samples were added to 4x loading buffer, vortexed, and boiled before immunoblotting and Coomassie staining.

[0241] RGDL probe dosing on live cells for immunoblot analysis: Wildtype U87-MG cells were seeded in 12-well plates at 250,000 cells per well in 1.5 mL of RPMI media (CORNING®) supplemented with 10% fetal bovine serum (CORNING®) and 1% penicillin / streptomycin (GIBCO®) 24 hours before the experiment. Cells were treated with 0, 0.5, 1, 5, 10 and 25 pM of RGDL in 500 pL of serum-free, phenol-red-free RPMI media (GIBCO®) for 1 h at 37 °C. After incubation, media was replaced with 500 pL of PBS with RGDL (0-25 pM) and BP (250 pM). The cells were irradiated at 440 nm under an LED (PR160L, KESSIL®) for 1 min on ice. The cells were then washed in 1 mL of PBS twice and lysed in ice-cold lx RIPA buffer supplemented with protease inhibitors (ROCHE®). Cells were lysed while shaking on ice for 20 min before transfer and tip sonication (3 x 1 s pulses at 30% amplitude). The lysates were collected and processed for anti-biotin immunoblot analysis.

[0242] For comparative experiments between high and low integrin expressing cell lines, U87-MG and HEK293T cells were each seeded in 12-well plates at 250,000 cells in 1.5 mL of RPMI 48 h and 24 h before experimentation for U87-MG and HEK293T, respectively. Cells were treated with 5 and 10 pM of RGDL in 500 pL of serum-free, phenol-red-free RPMI media for 1 h at 37 °C. Cells were then irradiated and processed as indicated above. Lysates were then normalized to 1 mg / mL using BCA Protein Assay Kit (PIERCE®) and processed for antibiotin immunoblot analysis. Total lane intensities were quantified in Image J and normalizedto the most intense lane. Statistical comparison of intensities between each dose of probe between cells lines was performed in Prism (GRAPHPAD®) using multiple unpaired t-test.

[0243] SILAC cell culture methods and proteomic sample preparation: SILAC labeling was performed by growing cells for at least five passages in lysine- and arginine-free SILAC medium (RPMI or DMEM, INVITROGEN®) supplemented with 10% dialyzed fetal calf serum (GEMINI®) and 1% Pen / Strep. “Light” and “heavy” media were supplemented with natural lysine and arginine (0.1 mg / mL) for “light”, and 13C-, 15N-labeled lysine and arginine (0.1 mg / mL) for “heavy”, respectively.

[0244] Sample preparation and streptavidin enrichment: Quantitative proximity labeling studies with SILAC quantitative proteomics were performed with “heavy” and “light” labeled U87-MG, HeLa, MDA-MB-231, and BM1 cell lines. SILAC-labeled cells, grown to 80-90% confluency in 10 cm cell-culture treated plates each, were incubated with DMSO alone (light cells) or RGDL probe (50 pM, heavy cells) for 1 h in serum-free SILAC RPMI. After incubation, excess probe was removed by aspiration and supplanted with BP (250 pM) in DPBS. Cells were then irradiated at 440 nm using an LED (PR160L, KESSIL®) for 5 min, rinsed, scraped, and washed with cold PBS (1 ml x 4). The cells were pelleted and then lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% polyethylene glycol tert-octylphenyl ether (e.g., TRITON™ X-100), 0.5% deoxycholate, pH 7.4) supplemented with EDTA-free complete protease inhibitor (ROCHE®) and 1 mM DTT, at 4 °C. After sonication, insoluble debris was cleared by centrifugation (17,000 g, 15 min). BCA assay was performed to normalize Heavy and Light protein concentrations to ~1 mg / ml. Streptavidin agarose beads (50 pL slurry, PIERCE®) were washed twice with RIPA buffer, and each cell lysate was separately incubated with the beads with rotation overnight at 4 °C. The beads were subsequently washed five times with 0.5 mL of RIPA lysis buffer containing 1 mM DTT, combined together, then washed once with 1 mL of 1 M KC1, four times with 0.5 mL PBS, and two times with 2 M Urea in 25 mM ammonium bicarbonate. 500 pL of 6 M Urea in 50 mM ammonium bicarbonate was then added to the beads, and samples were reduced on resin by TCEP (10 mM final) with orbital shaking for 20 minutes at 65 °C. Samples were then alkylated by adding iodoacetamide (20 mM final), covered from the light and with orbital shaking, for 40 minutes at 37 °C. The streptavidin agarose beads were collected, washed once with 2 M Urea in 25 mM ammonium bicarbonate, and the buffer exchanged to 2 M Urea in 25 mM ammonium bicarbonate supplemented with 1 mM CaCh. Enriched proteins were digested on-bead by the incubation of 2 pg sequencing-grade trypsin overnight at 37 °C. Following trypsinization, the supernatant was collected, acidified with HPLC grade formic acid (2% final, pH 2-3), and peptides werethen desalted on ZipTip C18 tips (100 pL, MILLIPORE®), dried under vacuum, resuspended with LC-MS grade water (SIGMA ALDRICH®), and then lyophilized. Lyophilized peptides were dissolved in LC-MS / MS Buffer (H2O with 0.1% formic acid, LC-MS grade, SIGMA ALDRICH®) for proteomic analysis.

[0245] Cell detachment studies: U87-MG and HeLa cells were grown on 10 cm dishes to -80-90% confluency, rinsed with PBS and incubated with 5 mL of a non-enzymatic dissociation buffer (Cell Stripper, CORNING®) at 37 °C for 10 min and transferred to conical tubes (e.g., FALCON® tubes). Cells were then pelleted (300 g) at room temperature and washed once with serum -free RPMI (without phenol red). Cells were then resuspended in RGDL-probe containing media (50 pM) or DMSO for 1 h in 4 mL of SILAC media, followed by replacement with DPBS containing BP substrate (250 pM). Cells in suspension were then irradiated and processed as indicated above.

[0246] Mouse Xenograft Studies: All animal protocols related to mouse experiments were approved by the University of Chicago’s Institutional Animal Care and Use Committee. Approximately 1 x 106MDA-MB-231 cells in 100 pL of PBS were injected into the fourth mammary fat pad of 8-10-week-old female athymic nude mice (CHARLES RIVER®). Approximately 2.5 x 106U87-MG cells in 100 pL of 1 : 1 PBS / Matrigel (phenol red-free, CORNING®) were injected subcutaneously on the lower left flank of 16-week-old male athymic nude mice (CHARLES RIVER®). When tumors reached approximately 500 mm3, mice were sacrificed, and tumors were dissected. The tumor samples were then gently diced, rinsed in DPBS, and incubated in RGDL (50 pM) in phenol-red-free RPMI media or vehicle for 1 hour with rotation at room temperature. Excess probe was removed, and the samples were then treated with BP (250 pM) in DPBS solution and irradiated at 440 nm for 1 or 5 min over ice. After irradiation, tissue samples were washed 4 times with 1 mL DPBS. Tissues were then treated with 1 x RIPA buffer with protease inhibitor and tip sonicated over ice. Lysates were then clarified and subjected to streptavidin enrichment and proteomic sample preparation as indicated.

[0247] Whole proteome LC-MS / MS analysis of TNBC cells: MDA-MB-231 and BMl cells were grown to confluence in 6 cm dishes before scraping and pelleting. Cells were washed with DPBS twice (1 mL) before resuspension in 4 M Urea and 50 mM ammonium bicarbonate with protease inhibitors. Cells were then lysed via tip sonication over ice. Lysates (500 pL, each normalized to 1 mg / ml) were then treated with TCEP (10 mM final) and heated to 65 °C for 20 min. Samples were then cooled to room temperature before the addition of iodoacetamide (15 mM final) and incubation at room temperature for 30 min in the dark.Lysates were then diluted by adding a like volume of DPBS and CaCh (1 mM final). Sequencing grade trypsin was then added (1 :50 trypsimprotein) at 37 °C overnight. Peptide digest reactions were then stopped by cooling to room temperature and the addition of formic acid (2% final). Peptides were then desalted as indicated above. Peptides were then reconstituted in LC-MS grade water (0.1% Formic Acid, OPTIMA®) and normalized using a Quantitative Colorimetric Peptide Assay (PIERCE®) before analysis by LC-MS / MS.

[0248] LC-MS / MS Acquisition and Analysis: The proteomic methods reported were adopted from previous reports (Speltz et al., 2023). LC-MS / MS analysis for proteomics samples was performed with an UltiMate 3000 RSLCnano System (THERMO FISHER SCIENTIFIC®) using an Acclaim PepMap RSLC Cl 8 column (75 pm x 15 cm, 2 pm, 100 A, THERMO FISHER SCIENTIFIC®) with an in-line Acclaim PepMap 100 Cl 8 trap column (75 pm x 2 cm, 3 pm, 100 A, THERMO FISHER SCIENTIFIC®) heated to 45 °C. The LC system was coupled to an Orbitrap Exploris 480 and Nanospray Flex Ion Source with stainless steel emitter tip (THERMO FISHER SCIENTIFIC®). Mobile phase A was composed of H2O supplemented with 0.1% formic acid, and mobile phase B was composed of CH3CN supplemented with 0.1% formic acid. The instrument was run at 0.3 pL min'1with 2 h gradients. MS / MS spectra were collected for the entirety of the gradient using a data- dependent, 2-second cycle time setting with the following details: full MS scans were acquired at a resolution of 120,000, scan range of 380 m / z to 1,500 m / z, maximum IT of 25 ms, normalized AGC target of 300% and data collection in profile mode. MS2 scans were performed by high-energy collision dissociation (HCD) fragmentation with a resolution of 15,000, normalized AGC target of 50%, maximum IT of 50 ms, HCD collision energy of 30% and data collection in centroid mode. The isolation window for precursor ions was set to 1.6 m / z. Peptides with a charge state of 1, 7+ and unassigned were excluded, and dynamic exclusion was set to 40 seconds. The RF lens % was set to 40 with a spray voltage value of 2.0 kV and an ionization chamber temperature of 300 °C.

[0249] Data were processed using the SEQUEST HT search engine node within the Proteome Discoverer 3.0 software package. Data were searched using a concatenated target / decoy UniProt database of the human proteome with isoforms. Digest enzyme specificity was set to trypsin with up to two missed cleavages allowed, and peptide length was set to between 6 and 144 residues. Precursor mass range was set to 350-6500. Precursor mass tolerance was set to 10 ppm, and fragment mass tolerance was set to 0.02 Da. Up to 4 dynamic modifications were allowed per peptide, including heavy lysine (+8.0142), heavy arginine (+10.0083), oxidized methionine (+15.9949), N-terminal acetylation (+42.0106), N-terminalMet-loss (-131.0405) and N-terminal Met-loss + acetylation (-89.0299). Cysteine carboxyamidomethylation (+57.0215) was set as a static modification. A minimum of two peptides, with a minimum length of 6, was required for protein identification, and false discovery rate (FDR) was determined using Percolator with FDR rate set at 1%. Before quantification, chromatographic alignment was performed, with a maximum retention time difference of 10 min allowed, a mass tolerance of 10 ppm and a minimum signal / noise threshold of 5 required for feature mapping. SILAC ratios were determined using precursorbased quantification in a pairwise manner based on peak intensity without normalization or scaling using a maximum ratio of 20. For RGDL-PhotoPPI studies using SILAC for quantitation, proteins considered as enriched interactors were detected and quantified in at least 2 biological replicates and exhibited an RGDL-dependent median SILAC ratio greater than 4. MS files from LFQ samples were analyzed using the processing method parameters as indicated above with no dynamic modifications for heavy lysine or arginine. For whole proteome analysis of MDA-MB-231 and BM1 cells, a normalized pairwise ratio of BM1 over MDA-MB-231 was calculated in Proteome Discoverer. Proteins were considered significantly higher expressed if they displayed a fold change of at least 2 with a p-value less than 0.05. For RGDL experiments on tissue samples, abundance ratios of ‘probe’ over ‘no-probe’ were generated using Perseus accompanied by unpaired t-test. Positively enriched proteins were selected with the criteria of containing at least 4 peptides in the RGDL treated sample and a log2(ratio) greater than or equal to 4 with a p-value less than 0.05.

[0250] StringDB version 11.5 was used to find known PPIs among the enriched proteins (Szklarczyk et al., 2015). Physical networks from PPIs with experimental evidence from literature and databases using medium confidence (interaction score of at least 0.400) were selected in StringDB searches. Scatter plots (referred to as “Yang plots” in the text) showing the relationship between known PPIs and grouped ion intensity of enriched proteins were generated from the node degrees data from STRING analyses (Y-axis values) and the median grouped ion intensity (X-axis values) across biological replicates within the experiment at hand. Z-scores for the enriched proteins were calculated using the median grouped ion intensity.

[0251] Gene ontology and pathway analyses: Enriched proteins that are members of the GO term ‘Integrin Binding’ were used to generate integrin signaling sub-networks (Harris et al., 2004; Subramanian et al., 2005). Metascape analyses were performed on enriched proteins to identify significantly enriched gene sets and pathways (Zhou et al., 2019). Gene sets from GO Molecular Functions, GO Biological Processes, Hallmark Gene Sets and KEGG Pathwaywere used for Metascape queries (Harris et al., 2004; Liberzon et al., 2015; Kanehisa et al., 2000; Kanehisa et al., 2023). Enrichment P-values from the Metascape analyses were used to generate radar plots and heatmaps to compare differences and similarities between sample types. In addition, some significantly enriched pathways were selected for detailed heatmaps showing abundance ratios of proteins belonging to the respective pathway. Heatmaps were generated using Morpheus software (Broad Institute).

[0252] Principal component analyses of abundance ratios of enriched proteins: Principal component analyses (PCA) were performed using the parallel analysis method within Prism 10 software (GRAPHPAD®). Briefly, protein abundance ratios from biological replicates were used as input data for PCA analysis. The data were centered to have a mean of 0, and principal components with eigenvalues greater than the 95th percentile of the eigenvalues from 1000 simulations were selected. PCA loading plots showing PCI and PC2 of every biological replicate were generated using Prism.

[0253] Differential enrichment analyses of MDA-MB-231 and BM1 cells: Identified proteins from MDA-MB-231 and BM1 with positive log2(ratio) values were considered to compare differential enrichment between the two cell lines. A value of 0 was then assigned using imputation for missing log2(ratio) values. T-tests (two tailed, unpaired) on log2(ratios) of four biological replicates were performed in Perseus software to identify proteins with significantly different enrichment between the two cell lines (P -value < 0.01). Proteins with at least a 2-fold ratio difference were considered as higher enriched in one cell line over the other. For uniquely enriched proteins with a maximum ratio in each biological replicate, -logio(P- value) was uniformly set to 10. -Logio(P -value) from T-tests and log2(ratioBMi / ratio23i) were used to generate a volcano plot.

[0254] Metascape analyses were then performed on these two groups: unique or higher enriched proteins in MDA-MB-231 cells (“231 high”) or BM1 cells (“BM1 high”).

[0255] The Proximity Score of a protein was calculated using the formula: Proximity Score = log2(abundance ratio) * logio(“Heavy” ion intensity). For each protein, a Proximity Score was calculated for every biological replicate using its respective abundance ratio and “heavy” ion intensity. Proteins with an abundance ratio less than 4 in both cell lines (i.e., not enriched in the RGDL experiment) were assigned a Proximity Score of 0.

[0256] Survival analyses o f breast cancer patients: Genes from proteins selected from the integrin interactome profiling data were used for patient survival analyses. A 20-gene signature was selected from MDA-MB-231 cells or BM1 cells based on the significant and unique enrichment of the target in either the 231 or BM1 RGDL-treated sample, high median ionintensity within that enriched target group and belonging to plasma membrane or cell surface gene ontology groups. For inclusion in the mRNA expression analysis the integrin-interacting target must have been covered by the microarray probe set used in the existing patient survival dataset. Interaction barcode protein lists were identified as discussed in Example 3 and in FIG. 9; each gene list was used to query relapse-free survival data of TNBC patients from a microarray -based dataset or overall survival data of breast cancer patients without filtering of subtypes from an RNA-seq-based dataset (Gyorffy et al., 2021). TNBC patients were identified by all negative results in ER status by IHC, PR status by IHC, and ERBB2 status by microarray. Patient survival analyses were performed using the KM-plotter software (Gyorffy et al., 2024). Patients were grouped by average expression levels across each 20-gene signature, and the high and low-expression cutoffs were determined by KM-plotter by selecting the cutoff resulting in the highest statistical significance in the logrank test by p-value.II. Example 2. Chemical SynthesisA. Chemical synthesis of RGDL photoproximity probe (FIGs. 11-12).

[0257] Dinitrobenzoic Acid: To a dried round bottom flask, 7.2 g (40 mmol) of 2-Methyl- 4-nitrobenzoic acid (OAKWOOD CHEMICAL ®) was added. Concentrated sulfuric acid (40 mL) was then added and cooled to 0 °C. Next, concentrated nitric acid (40 mL) was added dropwise and the reaction was allowed to gradually reach room temperature over the course of an hour. The reaction was allowed to stir for 24 hr before cooling to 0 °C and adding cold di to further precipitate product. The mixture was allowed to stir thoroughly before filtering the solid and rinsing thoroughly with cold di and hexanes. After drying via vacuum, 2-methyl-4,5- dinitrobenzoic acid (1) remained as a bright yellow-orange solid (7.9 g, 88% yield).1H NMR (500 MHz, DMSO-d6) 5 8.51 (s, 1H), 8.23 (s, 1H), 2.69 (s, 3H).

[0258] Photocatalyst 2 (PC 2): Intermediate 1 (4 g, 1 Eq.) was then dissolved in neat ethanol (35 ml, 0.5 M). SnCh 2H2O (24 g, 6 Eq.) was added portion wise and the reaction was heated to 80 °C. The reaction was stirred for 2 h before being cooled to room temperature and added to cold di. The suspension was then basified with 1 M NaOH to pH 7 at 0 °C and allowed to stir for 1 hr. Acetic acid was then added dropwise to attain a pH of 5. The resulting orange precipitate was then filtered and the flow through was put on vacuum in order to remove excess alcohol. Next, the solution was extracted thoroughly with EtOAc. Combined organics were then washed with brine and dried with sodium sulfate. Solvents were removed and this material was put through a silica plug using EtOAc as an eluent. Following removal of the solvent, theresulting orange solid was used in the next step without further purification (930 mg, 32% crude yield). This solid (1 Eq.) was added to a dried round bottom flask and dissolved in acetic acid (28 mL, 0.15 M). Next, boric acid (SIGMA®, 342 mg, 1 Eq.) and alloxan monohydrate (SIGMA®, 885 mg, 1 Eq.) were added and allowed to stir for 2 hr. at room temperature. After this time, a solid precipitated from solution and was then filtered. The yellow solid was washed thoroughly with cold acetic acid and then ether. The solid was then collected and triturated in water and a solution of EtOAc and ether (50:50). Removal of solvents via vacuum afforded lumichrome benzoic acid (2) as a yellow solid (1.29 g, 86% yield).JH NMR (500 MHz, DMSO-d6) 5 12.05 (d, 1H), 11.78 (s, 1H), 8.52 (s, 1H), 8.23 (s, 1H), 8.07 (s, 1H), 7.80 (s, 1H), 2.74 (d, 3H).

[0259] Lumichrome-NHS (1): Photocatalyst 2 (295 mg, 1 Eq.) was then added to a dried round bottom flask and dissolved in DMF (3.6 ml, 0.3 M). NHS (218 mg, 1.75 Eq.) and EDCI (362 mg, 1.75 Eq.) were added and the contents were stirred for 3 h at room temperature. Solvents were then removed using vacuum before addition of 3.5 ml of di to precipitate yellow solid. The suspension was allowed to mix thoroughly before filtration. The solid was rinsed thoroughly with cold di and then cold ether before being dried by vacuum to yield lumichrome- NHS (3) as a bright yellow solid (211 mg, 53% yield). 'H NMR (400 MHz, DMSO-d6) 3 12.10 (s, 1H), 11.85 (s, 1H), 8.73 (s, 1H), 8.42 (s, 1H), 8.25 (s, 1H), 7.96 (s, 1H), 2.94 (s, 4H), 2.75 (d, 3H).

[0260] RGDL Probe: 90 mg of Fmoc-Asp(OtBu) loaded Wang resin (ADVANCED CHEMTECH®, 100-200 mesh, 0.6 mmol / g) was swelled in DMF for 30 minutes and deprotected in 20% piperidine / DMF solution (2x10 min). Following standard Fmoc procedure, the subsequent Gly and Arg residues were coupled to the resin (10 eq AA, 9.9 eq HCTU, 20 eq DIPEA). An Fmoc-6-aminohexanoic acid linker was coupled following the same procedure. The N-terminal amine was deprotected in 20% piperidine / DMF, then lumichrome-NHS (54 pmol) was added in DMF, with addition of 19 pL DIPEA, and was coupled to the peptide. The peptide was cleaved from the resin using 2 mL of a 95:2.5:2.5 TFA / H2O / TIS cocktail for 2 hours, then precipitated with the addition of 20 mL cold ethyl ether. Peptide was pelleted (4696 x g, 5 min) and resuspended in 2 mL H2O, and purified by HPLC-MS to yield RGDL as a bright yellow solid (24.7 mg, 64% yield), LC-MS (714 m / z, M + 1).B. Chemical Synthesis of RGDL substrate probes (FIGs. 13-25).

[0261] Nor biotinamine synthesis (FIG. 13): D-biotin (3 g, 1 Eq.) was added to an oven dried round bottom flask. DMF (12 mL) and tButOH (12 mL) were added to dissolve the solid. TEA (2.6 mL, 1.5 eq.) and DPPA (2.9 mL, 1.1 Eq.) were then added dropwise at room temperature. The contents were then heated to a temperature of 70 °C and allowed to stir overnight. 1 M NaOH (25 mL) was then added dropwise over ice resulting in the evolution of gas. The contents was then extracted thoroughly using EtOAc, washed with brine and dried with sodium sulfate. The organic solvents were removed and the residue was triturated in 5% MeOH in EtOAc before filtration of an off white powder yielding Boc-norbiotinamine (2.6 g, 68% yield). Bocnorbiotin (1.46 g) was then added to a round bottom flask and dissolved in 50 % TFA in DCM (23 mL, 0.2 M) and allowed to stir for 4 h at room temperature. Solvents were then removed using nitrogen inlet overnight followed by high vacuum the next morning. The residue was then dissolved in methanol and added to A-21 Amberlyst resin and allowed to stir for 30 min at room temperature to remove excess acid. The resin was then filtered and the solvents were then removed from filtrate. The amine was then triturated in EtOAc before filtration of norbiotinamine as an off-white solid (1.33 g, 87 % yield).JH NMR (500 MHz, DMSO-d6) d 6.47 (d, 2H), 4.34 (m, 1H), 4.16 (m, 1H), 3.13 (m, 1H), 2.86 (dd, 1H), 2.77 (m, 2H), 2.61 (d, 1H), 1.62 (m, 6H).

[0262] Substrate 1 (SI) synthesis (FIG. 14): To a dry round bottom flask, SDA (0.5 g, 1.0 Eq.), Boc-Lysine-OH (0.82 g, 1.5 Eq.) were added and dissolved in DMF (14 mL, 0.16 M). DIPEA (0.97 mL, 2.5 Eq.) was added and the reaction was allowed to stir overnight. The next day, di was added and the reaction was extracted thoroughly with Et2O and washed sparingly with di and brine. After evaporating the organic solvent, the resulting residue was purged with N2to yield Photo-Lys(Boc) as a yellow oil (0.735 g, 93% yield). 'H NMR (500 MHz, DMSO- d6) b 12.43 (s, 1H), 3.82 (m, 1H), 3.00 (m, 2H), 1.94 (t, 2H), 1.62 (m, 1H), 1.56 (t, 2H), 1.38 (s, 9H), 1.30 (m, 5H), 0.99 (s, 3H).

[0263] Photo-Lys(Boc) (0.735 g, 1 Eq.) was then taken up in DMF (7 mL, 0.3 M). NHS (0.356 g, 1.5 Eq.) and EDCI (0.592 g, 1.5 Eq.) were added and the reaction was allowed to stir overnight at room temperature. The next morning, di was added and the reaction was extracted thoroughly with Et2O three times. Combined organics were then washed with water, brine, and then dried over with Na2SO4. The residue was put on high vacuum and stored overnight at - 20 °C to yield Photo-Lys(Boc)-NHS ester as an off-white wax which was used without further purification (0.6415 g, 68% yield). Next, Photo-Lys(Boc)-NHS ester (297 mg, 1 Eq.) and norbiotinamine (423 mg, 3 Eq.) were added to a dried round bottom flask. The contents were then dissolved in DMF (6.5 mL, 0.1 M) and DIPEA (0.23 mL, 2 Eq.) was added. The contentswere allowed to stir at room temperature overnight. The next day, a mixture of dl / NT Cl was added to the reaction and the contents were extracted thoroughly with EtOAc. The combined organics were then washed with di and dried over Na2SO4. The residue was then purified by silica column chromatography using 1 / 30 MeOH / DCM followed by 1 / 10 MeOH / DCM to elute product. Rotary evaporation was used to remove all organic solvents revealing Photo- Lys(Boc)-biotin as a white solid (0.316 g, 87%). 'H NMR (500 MHz, DMSO-d6) 3 7.84 (t, 1H), 7.76 (t, 1H), 6.73 (d, 1H), 6.43 (d, 2H), 4.31 (m, 1H), 4.13 (m, 1H), 3.81 (m, 1H), 3.18 (d, 1H), 3.09 (m, 2H), 2.99 (m, 3H), 2.84 (dd, 1H), 2.60 (d, 1H), 1.94 (t, 2H), 1.61 (m, 1H), 1.56 (t, 2H), 1.38 (s, 9H), 1.24 (m), 0.98 (s, 3H).

[0264] Photo-Lys(Boc)-biotin (0.316 g) was taken up in a mixture of TFA / DCM (2: 1, 6 mL, 0.1 M) and allowed to stir for 3 hr. at room temperature. N2 was then used to remove all solvents overnight. The residue was then dissolved in MeOH and added to Amberlyst A21 resin to stir for 30 min to remove excess acid. The resin was then filtered, and solvents were evaporated. The contents were then subjected to high vacuum to yield Substrate 1 (SI) as a white solid (0.238 g, 92% yield). 'H NMR (500 MHz, DMSO-d6) 3 7.85 (s, 2H), 6.46 (d, 2H), 4.33 (m, 1H), 4.15 (m, 1H), 3.12 (m, 7H) 2.84 (dd, 1H), 2.57 (d, 1H), 1.94 (t, 2H), 1.56 (t, 2H), 1.43 (m, 12H), 0.99 (s, 3H). LC-MS (454 m / z, M+l).

[0265] Substrate 2 (S2) synthesis (FIG. 15): 4-[3-(Trifluoromethyl)-3J / -diazirin-3- yl]benzyl alcohol (TCI CHEMICALS®, 28 mg, 1 Eq.) was added to an oven dried 2-dram scintillation vial. DSC (53 mg, 1.6 Eq.) was added followed by the addition of acetonitrile (0.65 mL, 0.2 M). TEA (0.058 mL, 3.22 Eq.) was then added dropwise and the contents were allowed to stir for several hours at room temperature. Norbiotinamine (43 mg, 1 Eq.) was then dissolved in acetonitrile (0.65 mL) and DMF (0.2 mL). This solution was then added to the reaction and contents were allowed to stir at room temperature overnight. Solvents were then removed and 1 mL of di was added before trituration and filtration. The solids were then triturated in a solution of 1% MeOH in EtOAc in ether before filtration to yield Substrate 2 (S2) as a white solid (18 mg, 30%)XH NMR (500 MHz, DMSO-d6) 3 7.46 (d, 2H), 7.29 (d, 2H), 6.40 (d, 2H), 5.03 (s, 2H), 4.30 (m, 1H), 4.10 (m, 1H), 3.08 (m, 1H), 2.98 (m, 2H), 2.82 (dd, 1H), 2.57 (d, 1H), 1.59 (m, 1H), 1.43 (m, 3H), 1.31 (2 H).19F NMR (500 MHz, DMSO-d6) 3 -64.66 (s). LCMS (458 m / z, M+l).

[0266] Substrate 3 (S3) synthesis (FIG. 16): SDA (30 mg, 1 Eq.) and norbiotinamine (65 mg, 1.5 Eq.) were added to an oven dried 2-dram scintillation vial. DMF (0.53 mL, 0.25 M) was then added, followed by the addition of DIPEA (60 pL, 2.5 Eq.) and allowed to stir overnight at room temperature. Solvents were then removed before the addition of 1 mL ofcold di. The contents were stirred thoroughly before filtration. The solid contents were then dissolved in methanol and purified by silica column chromatography using 1 / 10 Methanol / DCM. Solvents were then removed yielding Substrate 3 (S3) as a solid (12.9 mg, 29 % yield). 'H NMR (500 MHz, DMSO-d6) 3 7.81 (1H), 6.40 (d, 2H), 4.30 (m, 1H), 4.12 (m, 1H), 3.08 (m, 1H), 3.00 (m, 2H), 2.92 (dd, 1H), 2.57 (d, 1H), 1.94 (t, 2H), 1.60 (m, 1H), 1.56 (t, 2H), 1.39 (m, 5H), 0.96 (s, 3H). LC-MS (326 m / z, M+l).

[0267] Substrate 4 (S4) synthesis (FIG. 17): Aminocaproic acid (1 g) was added portion wise to TFAA (4 mL) in a dried round bottom flask and allowed to stir at room temperature for several hours. A solution of di and NH4Q was added and the organics extracted with ether. The organics were washed with brine and dried with Na2SO4. The organic solvent was then removed to yield a white powder (1.3 g, 75 % yield) which was used without further purification. The carboxylic acid (1.2 g, 1 Eq.) was then added to a dried round bottom flask. NHS (0.92 g, 1.5 Eq.) and EDCI (1.53 g, 1.5 Eq.) were then added before dissolution in DMF (18 mL, 0.3 M). The contents were allowed to stir overnight before organic extraction using ether. The organics were then washed with sodium bicarbonate twice, brine, and dried using Na2SO4. Removal of organic solvent yielded Trifluoroacetamide-C6-NHS ester as a thick oil (1.38 g, 79 % yield) 'H NMR (500 MHz, DMSO-d6) 3 6.99 (s, 1H), 3.40 (q, 2H), 2.85 (4H), 2.63 (t, 2H), 1.80 (m, 2H), 1.64 (m, 2H), 1.45 (m, 2H).

[0268] NHS ester 7 (0.25 g, 1 Eq.) and norbiotinamine (0.306 g, 1.2 Eq.) were added to a dried round bottom flask. The contents were dissolved in DMF (3 mL, 0.25 M) and allowed to stir overnight before the addition of cold di (10 mL) resulting in precipitation of a solid. The contents were mixed thoroughly before filtration of Trifluoroacetamide-C6-biotin as a white solid (305 mg, 93% yield). 'H NMR (500 MHz, DMSO-d6) 5 9.40 (s, 1H), 7.74 (s, 1H), 6.43 (d, 2H), 4.31 (m, 1H), 4.14 (m, 1H), 3.19 (m, 2H), 3.01 (m, 3H), 2.74 (dd, 1H), 2.60 (d, 1H), 2.05 (t, 2H), 1.44 (m, 12H).

[0269] Intermediate 8 (0.27 g, 1 Eq.) and K2CO3 (0.44 g, 5 Eq.) were then added to a dried round bottom flask. A solution of water / methanol (20 / 1, 3.2 mL) was added and the contents were refluxed for 3 h. The contents were cooled to room temperature and the K2CO3 filtered. d!-H2O was added and the contents were acidified with 12 N HC1 to pH 6-7. Solvents were then removed using vacuum yielding amine as an amorphous solid (297 mg) which was used without further purification (LC-MS, 329 m / z, M+l). Next, SDA (95 mg, 1 Eq.) was added to a dried round bottom flask followed by addition of the amine (209 mg, 1.5 Eq.) dissolved in DMF (2 mL). DIPEA (110 pL, 1.5 Eq.) was then added and the contents were stirred at room temperature for 1 h. The solvents were then removed before trituration in 2 mL of cold dI-H2O.Following removal of solvents, the contents were dissolved in methanol and purified by HPLC. Solvents were removed and the resulting residue was treated with cold ether. The resulting precipitate was filtered to yield Substrate 4 (S4) as a white solid (70 mg, 38% yield).JH NMR (500 MHz, DMSO-d6) 5 7.79 (s, 1H), 7.70 (s, 1H), 6.40 (s, 1H), 6.33 (s, 1H), 4.30 (m, 1H), 4.12 (m, 1H), 3.09 (m, 1H), 3.00 (m, 4H), 2.82 (dd, 1H), 2.57 (d, 1H), 2.01 (t, 2H), 1.91 (t, 2H), 1.62 (m, 1H), 1.54 (t, 2H), 1.45 (m, 11H), 0.96 (s, 3H). (LC-MS, 439 m / z, M+l).III. Example 3 - Family-Wide Photoproximity Profiling Identifies Disease-Associated Integrin Protein Social NetworksA. Abstract

[0270] Integrin family transmembrane receptors mediate dynamic interactions between cells and their extracellular microenvironment. The heterogeneous interaction partners of integrins directly regulate cell adhesion, motility, proliferation, and intracellular signaling. Despite the recognized importance of protein-protein interactions and the formation of signaling hubs around integrins, the ability to detect and quantify these dynamic binding partners with high spatial and temporal resolution remains challenging. Here, the inventors developed an integrin family-directed photocatalytic chemical probe and quantitative photoproximity proteomic profiling method to detect and quantify native integrin-centered protein social networks on live cells and tissues without the need for genetic manipulation, antibodies, or non-physiologic cell culture conditions. The inventors drafted quantitative maps of integrin-centered social networks, highlighting conserved and unique binding partners between different cell types and cellular microenvironments. Quantitative comparison of integrin social networks in cancer cell lines of diverse tissue of origin and disease state identified specific AND-gate binding partners that served as markers for tumor cell metastatic state. For example, in metastatic triple negative cancer (TNBC) cell lines the inventors identified the presence of differentiated integrin complexes with certain known and novel interaction partners involved in cell migration, metastasis and proliferation. These individual members represented AND-gate markers in pre- and post-metastatic TNBC cell populations. Finally, the expression of integrated social network barcodes generated from these profiles strongly correlated with both positive and negative disease progression and outcomes in TNBC patients. Taken together, these data provided the first family-wide high-resolution maps of native protein interactors on live cells and identified dynamic integrin-centered social networks as potential AND-gate markers of cell identity, microenvironmental context, and disease state.B. Introduction

[0271] The cell surface proteome is a dynamic and heterogeneous landscape of proteins involved in myriad cellular processes. Among the many classes of proteins present on the cell surface, a subset is directly involved in communicating signals to and from neighboring cells and extracellular matrix components, thereby regulating intrinsic cellular responses to the extracellular environment. The integrin family of cell surface proteins is comprised of 18 distinct a-integrins and eight P-integrins, which form a network of 24 known functional heterodimers (Humphries et al., 2006; Humphries et al., 2000). These transmembrane complexes mediate bidirectional, ‘inside-out’ and ‘outside-in’ signaling. ‘Outside-in’ signaling is typically induced by extracellular ligand binding, which facilitates cell spreading, retraction, migration, and proliferation, among other processes. Meanwhile, ‘inside-out’ signaling events are propagated by integrin cytoplasmic domains, which ultimately activate ligand binding and adhesiveness, as well as cellular migration (Hamidi et al., 2018; Hood et al., 2002; Li et al., 2023). Integrin heterodimer complexes are subclassified depending on their known ligand associations, including collagen receptors, laminin receptors, leukocyte-specific receptors, and ‘RGD’ receptors (Mezu-Ndubuisi et al., 2021; Takada et al., 2007; Hynes et al., 2002). In cancer, integrins are implicated in several aspects of disease progression. The increased expression of integrins and activation of integrin signaling pathways contribute to proliferative phenotypes, local invasion and intravasation of metastasizing cancer cells, and survival of circulating tumor cells. Altered integrin activity is also implicated in the colonization, survival, and therapeutic resistance of metastatic tumor colonies (Hamidi et al., 2018; Cooper et al., 2019; Seguin et al., 2015; Parvani et al., 2013). Despite the strong correlation between dysregulated integrin levels in cancer, relatively little is known about the protein-complex membership in integrin-centered ‘migratory hubs’ involved in bidirectional signaling with extracellular environments.

[0272] Previous studies have explored integrin interactors and signaling using a variety of canonical methods, including fluorescence microscopy, immunofluorescence, gene silencing, and affinity purification-mass spectrometry (AP-MS), amongst others (Morello et al., 2011). These studies have identified tissue- and disease-specific integrin complex members (Mandal et al., 2023), as well as context-dependent interaction partners, including receptor tyrosine kinases like c-Src and EGFR (Moro et al., 2002; Alfonzo-Mendez et al., 2022). However, few chemical tools or methods exist for quantitative proteomic profiling of native integrins and integrin protein-protein interactions (PPIs) in live cells and tissues, mainly because quantitativedetection of dynamic cell surface protein interactomes remains a significant technical challenge. Traditional AP-MS or immunoprecipitation approaches can overlook low affinity or transient protein interactions, fail to capture in situ protein interaction dynamics and raise the potential for false positive detection of interactions under non-physiologic lysis and pulldown conditions, among other challenges.

[0273] The inventors hypothesized that many technical limitations could be overcome by the application of in situ ‘proximity profiling’ methods, which utilize locally activated reactive chemical tagging agents to label direct and proximal protein interactors (Huang et al., 2020; Trinkle-Mulcahy et al., 2019; Lundberg et al., 2019; Qin et al., 2021; Seath et al., 2021). Enzymatic proximity profiling methods such as BioID, APEX, and PUP-IT rely on the expression of genetic fusions to a specific protein of interest, resulting in proximal binding partner labeling with reactive and retrievable chemical tags (Hung et al., 2014; Liu et al., 2018; Roux et al., 2012; Lee et al., 2016). More recently, the inventors described a light-activated proximity profiling approach, PhotoPPI, which enables mapping of intracellular and extracellular protein interactors through the local activation of reactive carbenes in response to a light trigger (Huang et al., 2020; McCutcheon et al., 2020). This work, as well as subsequent approaches that use antibody conjugates of Ir- or small molecule-photocatalysts (Geri et al., 2020; Lin et al., 2024; Seath et al., 2023) demonstrate the potential for minimally perturbative, rapid, and spatially restricted detection of protein interactors in or on live cells. The inventors reasoned that an ideal approach to profile native integrin ‘social networks’ would be to develop a similar technology without the need for genetic fusions, antibodies, or large-transition metal photocatalysts. To this end, the inventors developed a small molecule ligand-directed photoproximity method to map native and heterogeneous integrins and their interactors on live cells and tissues. The inventors used this novel method to detect cell- and context-dependent integrin ‘social networks’ that served as markers of cell state and were directly implicated in cancer pathology, such as patient survival in breast cancer.C. Results1. Design and Characterization of a Native Integrin-Directed Photoproximity Profiling Pipeline.

[0274] To develop a photoproximity protein interaction (PhotoPPI) profiling method capable of capturing the interaction partners of native integrins on live cells, the inventors first designed a fully synthetic chemical probe comprised of an ‘RGD’ peptide targeting motif,which is structurally similar to reported integrin-targeting agents (Liolios et al., 2021; Huang et al., 2012; Kapp et al., 2017), fused to a synthetic lumichrome photocatalyst (Chen et al., 2012; Tatsumi et al., 1992) (FIG. 1A). The RGD motif is a specific amino acid sequence naturally found on certain ECM proteins such as fibronectin, vitronectin, and laminin, which interacts with the RGD binding domains of more than half of all known integrins (Plow et al., 2000; Pierschbacher et al., 1984). Therefore, an RGD-containing chemical probe could localize efficiently to diverse integrin receptor complexes on cell surfaces and present an opportunity for ‘family-wide’ profiling of the proximal ‘social network’ of neighboring surface receptors and ECM proteins. This information could not be captured through the use of single protein target-specific antibody conjugates or expression of genetic fusions, which have been the predominant approaches for in situ proximity profiling methods (Huang et al., 2020; McCutcheon et al., 2020; Geri et al., 2020; Lin et al., 2024). To access a synthetically tractable and biologically compatible photocatalyst, the inventors first designed a novel amine-reactive lumichrome photosensitizer, 1, which should be broadly useful for conjugation to small molecules, peptides, and other biologies (FIG. 1A). The inventors chose lumichrome due to its photostability and ability to activate a range of groups in response to light to generate reactive species (Hung et al., 2014) for labeling of direct and proximal binding partners in situ. The inventors directly conjugated novel amine-reactive lumichrome photosensitizer 1 to the RGD- targeting peptide core on a solid-phase support, permitting direct cleavage and purification of the dual-function photoproximity probe, referred to hereafter as RGDL (FIG. 1A).

[0275] The inventor’s photoproximity profiling strategy aimed to localize RGDL molecules to native integrins on the cell surface where they can activate a spatially restricted, reactive substrate for proximal tagging of protein interactors and complexes (FIG. IB). This strategy relied on the co-localization of a suitably reactive substrate for activation in the presence of light for spatial and temporal control of labeling events. To test this premise, the inventors characterized the activation of a biotinamide phenolic substrate (referred to as BP) by the core photocatalyst, 2, in a model solution-labeling assay using BSA. The inventors observed strong BSA protein labeling at low micromolar concentrations of substrate (0-500 pm) and lumichrome photocatalyst (0-20 pM) in solutions exposed to either 365 or 440 nm light sources (FIGs. 1C-1D, and FIGs. 6A-6B). Photoactivation occurred rapidly (activation <1 min; FIG. ID) and with very low or undetectable background in the absence of light or photocatalyst. Other substrates, including reported trifluoromethyldiazirines, could be activated by lumichrome but exhibited some light-dependent activation in the absence of photocatalyst and were thus ignored for the purposes of the inventors (FIGs. 6C-6E). Next,the inventors performed experiments with RGDL, BP, and blue light activation directly on live U87-MG glioblastoma cells, demonstrating RGDL- and light-dependent biotinylation of native proteins under standard cell culture conditions (FIG. IE). The inventors next performed RGDL photolabeling on U87-MG and HEK293T cells, which represented relatively high- and low- integrin expressing cell lines, respectively (Taherian et al., 2011; https: / / www.proteinatlas.org / ENSG00000150093-ITGBl / cell+line;. https: / / www. proteinatlas. org / ENSG00000259207-ITGB3 / cell+line https: / / www.proteinatlas.org / ENSG00000138448-ITGAV / cell+line\ and observed significantly higher RGDL-dependent labeling in U87-MG cells (FIG. IF). Collectively, these data supported a rapid and integrin-dependent specific photoproximity labeling with RGDL and blue light, providing conditions for further quantitative exploration by mass spectrometrybased proteomic profiling on cells and tissues.2. Quantitative RGDL-PhotoPPI Profiling of Unique and Conserved Integrin Complexes on Live Cancer Cells.

[0276] Next, the inventors sought to identify integrin-centered protein interactors on live cells using an RGDL-directed, quantitative photoproximity proteomic workflow. The inventors first chose the U87-MG glioblastoma cells due to their relatively high expression of several RGD receptor integrins, such as ITGB1, ITGAV, ITGB3, as well as previous use of RGD- derived probes and imaging agents (Huang et al., 2012; Wang et al., 2013; Russo et al., 2013) with this cell line. Furthermore, despite being one of the most aggressive and common cancers that originate in the brain (Bleeker et al., 2012), little is known about integrin-dependent protein complexes and signaling pathways in glioblastoma. To detect and quantify RGDL-labeled, and therefore integrin-proximal target proteins, the inventors designed a PhotoPPI workflow using stable isotope labeling using amino acids in cell culture (SILAC) cell cultures. Matched U87- MG cultures were pre-incubated with RGDL (‘heavy’ cultures) or vehicle alone (Tight’ cultures), washed off of unbound ligand, and then added BP substrate followed by 440 nm light irradiation for 5 min (FIG. 2A). Under these conditions both cultures were exposed to substrate and light irradiation to control for any background, i.e., non-RGDL mediated protein detection in the workflow. After photolabeling, cells were lysed and processed for labeled protein enrichment and mass spectrometric analysis in a similar workflow previously developed for intracellular photoproximity profiling (McCutcheon et al., 2020).

[0277] Top enriched targets, both in terms of SILAC ratio (i.e., significant labeling in an RGDL-dependent manner) and median ion abundance were native integrins, including severalRGD-type receptor integrins such as ITGB1, ITGAV, ITGB3, and ITGA5. These top hits form known RGD-specific avP3 and a5pi heterodimeric receptors (FIGs. 2B-2C) (Kapp et al., 2017). In addition, the inventors were able to identify several laminin and collagen receptor integrins (ITGA1, ITGA2, ITGA11, ITGA3), presumably due to either direct probe interaction or close proximity to cell-surface signaling hubs containing RGD-binding integrins (FIGs. 2B- 2C). Beyond labeling core integrins, RGDL labeling detected numerous proteins that were known integrin-associated interactors, including several growth factor receptors and receptor tyrosine kinases such as EGFR (Petras et al., 2013), MET (Jahangiri et al., 2017), FGFR1 (Brown et al., 2016), AD AM-family cell surface proteases (Giebeler et al., 2016), as well as members of integrin-anchored focal adhesion complexes (e.g., VCL (Havugimana et al., 2012), PXL (Chen et al., 2000) and ACTN (Otey et al., 1990); FIGs. 2B-2C). To assess the relative strength or efficiency of integrin interactions beyond probe-dependent enrichment alone (i.e., SILAC ratio between probe and no-probe conditions), the inventors plotted the median ion intensity of enriched targets across biological replicates and performed an interaction-based STRING analysis of all enriched targets within the enriched target protein set from U87-MG cells (FIGs. 2C-2D). In the latter analysis, the inventors plotted the total known interactions for a given target protein with any other enriched protein in the dataset based on STRING databases from the literature (hereafter referred to as a “Yang plot”). The inventors observed that several core integrin members and known and novel associated surface receptors like EGFR, CD44, TGFB1, EPHA2, and MET were enriched in the upper right quadrant of the Yang Plot (FIG. 2C). Combined, the inventors posited that proteins demonstrating RGDL- dependent enrichment and significantly higher abundance and connectivity may represent core interactors with integrin complexes in these contexts.

[0278] With this enrichment workflow in place, the inventors next profiled the integrin social network in HeLa cells, a well-studied cervical cancer cell line with relatively lower expression of several integrin receptors such as ITGB1 and ITGB3. As in U87-MG cells, RGDL-PhotoPPI profiling identified several integrins (ITGB1, ITGA5, ITGA3, IGTB5 and others), surface receptors, proteases, and adhesion complex members (FIGs. 2D-2E; and FIG. 7A) as enriched and abundant complex members. Comparative analysis of enriched target proteins between U87-MG and HeLa cells revealed similarities and differences. Integrin ‘social networks’ on both cells contained proteins involved in cell-cell interactions, cadherin binding, and epithelial-mesenchymal-transition (EMT)-associated pathways (FIGs. 2E-2F). The quantitative nature of RGDL-PhotoPPI enabled the identification of distinct pathways and proteins across cell types. This included enrichment of neural crest and stem cell-relatedproteins in the glia-derived U87-MG cells, as well as increased enrichment of proteins involved in cell migration and angiogenesis. By contrast, less invasive HeLa cells contained more proximal proteins involved in endocytosis, protein folding, and specific cadherin family members (FIGs. 2E-2F). These comparative profiling analyses were congruent with previous work indicating that highly invasive U87-MG glioblastoma cells are dependent on integrin- mediated interactions, thus enabling development of diagnostic and therapeutic agents targeting integrins in this tumor type (MacDonald et al., 2001; Gladson et al., 1996). These data collectively suggested that RGDL-directed profiling on live cells can capture integrin interactors across various points in their lifecycle, including endocytosis, internalization, and perhaps receptor recycling (FIG. 2G) and that these interaction networks have unique membership associated with cell type and cell state.3. Cellular Environment Significantly Altered Integrin Social Network Membership.

[0279] Beyond comparing integrin social networks between disparate cell types, the inventors hypothesized that the flexibility of RGDL-dependent photoproximity profiling would permit interrogation of altered integrin interaction networks in response to changes in the extracellular environment. To test this hypothesis, the inventors probed the dynamic changes to integrin interaction wiring between adherent and detached U87-MG and HeLa cells, mirroring the populations of cells that are in contact with extracellular matrix versus those that have detached (i.e., undergoing metastasis or anoikis). While many studies have explored signaling changes or specific interactions involved in cell detachment and dissemination (Weems et al., 2023) little was known about the changes to integrin-centered interaction networks under these and many other context-dependent conditions.

[0280] Using matched adherent and detached cell cultures, the inventors performed RGDL-dependent photoproximity labeling, enrichment, mass spectrometry analysis, and quantitative comparison of interaction networks between each population (FIG. 3A). Principal component analysis confirmed clustering among cell type- and condition-matched replicates, which showed that altered extracellular environment had a more pronounced effect on the overall integrin social network than cell type (FIG. 3B). Integrins remained amongst the most abundant enriched target proteins in each cell line and under both adherent and detached conditions (FIGs. 3C-3E, and FIGs. 7A-7B). In addition to some integrins, there were interacting proteins that were equally enriched between adherent / detached cultures, such as ICAM5, but in general the core integrin-centered network and overall connectivity within these networks were reduced in detached U87-MG and HeLa cells (FIGs. 3D-3E). The inventorsperformed gene ontology (GO) analyses on detached and adherent cultures in both cell lines, which highlighted increased enrichment of ‘integrin binding’ and ‘receptor protein tyrosine kinase activity’ GO molecular function terms, among others, in adherent cells (FIG. 3F, and FIG. 7C). Similar analysis of enriched GO cellular component terms consistent with increased internalization of integrins (Tiwari et al., 2011), and a strong reduction in the enrichment of GO terms associated with cell-cell interaction, tight junctions, and extracellular matrix interactions in detached cells (FIG. 3F, and FIG. 7C). These data collectively demonstrated the ability of the RGDL platform to label and detect conserved and unique integrins, integrin PPIs, and integrin-dependent pathways across cancer cell lines of varied origin and microenvironmental context.4. RGDL-PhotoPPI Profiling on Native Tissue.

[0281] The inventors next sought to test whether the RGDL-PhotoPPI workflow was compatible with clinically relevant cell lines and primary tissues, starting with intact tumor tissues. Triple-negative breast cancer (MDA-MB-231) and glioblastoma (U87-MG) tumor xenografts were established in mice and excised upon reaching -300-500 mm3. Tumors were processed into smaller sections and processed for RGDL-PhotoPPI as described above for cell lines (FIG. 3G, and FIG. 8A). As with cell line studies, the inventors confirmed protein labeling only in the presence of RGDL probe on MDA-MB-231 tumors, as determined by Western blot, matching previous results with cell lines (FIG. 3G). The inventors then performed label-free quantification (LFQ) LC-MS / MS measurements following streptavidinbiotin enrichment to identify the integrin complex targets on U87-MG and MDA-MB-231 tumor tissues treated with 1 or 5 minutes of light, respectively (FIG. 3H, and FIGs. 8B-8C). Notably, the U87-MG-derived xenograft subjected to minimal irradiation resulted in robust enrichment of the primary integrin network (i.e., ITGB1, ITGB3) and established peripheral social network members (i.e., CD44, TGFBI, CAV1, CLTA, EGFR). Generally, experiments with both xenograft types resulted in a more robust enrichment of target integrins and integrin- binding PPIs, as compared with cell culture experiments. Furthermore, the tumor-derived integrin binding networks were highly interconnected and seemingly more complex (FIG. 31, and FIG. 8C); this may have been the result of greatly increased cell-cell interactions within the tissue matrix compared to two-dimensional cell cultures. Overall, these data confirmed that RGDL profiling can detect integrin-centered protein interactions on small (sub-mg) samples of primary tumor tissues and a facile workflow that could be applied to primary human tissues in future studies.5. Integrin Social Networks Correlated with Disease State and Outcomes.

[0282] The inventors next asked whether RGDL photoproximity profiling could identify interactions associated with disease states, such as cancer metastasis. The identification and characterization of surfaceome markers of metastasis, along with their use as prognostic markers or drug targets, could offer options for tailored therapy to improve patient outcomes. To determine how integrin social networks might change when cancer cells migrate from the primary site to a metastatic niche, the inventors performed quantitative RGDL profiling on two closely related triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 (231) and BM1.

[0283] The MDA-MB-231 cell line was developed from a pleural effusion collected from a TNBC patient. The metastatic BM1 cell line was developed by establishing MDA-MB-231 xenografts in immunodeficient mice, waiting for spontaneous metastasis and subsequent cell line generation from isolated bone metastases (Kang et al., 2003). RGDL profiling was performed with SILAC-labeled pairs of 231 and BM1 cell cultures as described for adherent cultures, followed by quantitative proteomics (FIG. 4A). While whole proteome profiling demonstrated relatively uniform integrin abundance between the two cell types (FIGs. 10A- 10B), RGDL interaction profiling identified 175 and 680 integrin interactors that were significantly increased in 231 and BM1 cells, respectively (FIGs. 4B-4C, and FIGs. 9A-9B). STRING bioinformatic analysis of enriched target proteins highlighted that core integrins (e.g., ITGB3, ITGA2, ITGAV, and ITGB1) and previously identified integrin interacting proteins (e.g., CD44, EGFR and MET) were shared in both cell lines. Likewise, most of the general pathways associated with enriched interactors were shared, including those related to migration, adhesion, proliferation, regulation of apoptosis, and angiogenesis (FIG. 9C). Within these networks there were many known and novel integrin-interacting proteins enriched. For example, ITGB3 and EGFR showed similar probe enrichment values (i.e., SILAC ratio) and integrated ion intensities in both 231 and BM1 cells. To try and capture integrin proximity and labeling efficiency, the inventors calculated the product of these two as an integrated 'proximity score,’ which likewise showed similar presence of ITGB3 and EGFR in both cell lines (FIG. 9D) Other known interactors were differentially enriched in the two cell lines. For example, VCAM1, a known integrin interactor (Humphries et al., 2009) and potential drug target in metastatic breast cancer (Chen et al., 2012) was highly enriched in 231 but not in post- metastatic BM1 cells (FIG. 4C), By contrast, ICAM2, a ligand of ITGB2 was only enriched in BM1 cells, which is not surprising given its implication as a promoter of brain metastasis and tumor cell sternness in TNBC (Pan et al., 2023). SLC3A2, a known interactor of ITGB1 (Rintoul et al., 2002), is overexpressed in many cancer types, including breast cancers, and hasbeen proposed as a drug target (El Ansari et al., 2018; Nachef et al., 2021). The inventors observed that SLC3A2 isoform 1 was uniquely enriched in 231 cells, while isoform 2 was found only in BM1 cells (FIG. 4C), highlighting isoform-specific complex tracking of cancer- associated targets with RGDL profiling.

[0284] Beyond known interactors, RGDL profiles highlighted many novel integrin interacting proteins that were differentially present in BM1 and 231 cells. For example, proteins related to angiogenesis were among the most significantly enriched integrin interactors in both cell lines (FIGs. 4D-4F). The angiogenic network was centered on an integrin core, with subnetworks including ephrin receptors and ROBO1 / SLIT signaling complexes. For example, while both cell lines show enrichment of neuropilins (NRP1 or NRP2), post-metastatic BM1 cells showed unique proximity between both ROBO1 receptor and its ligand SLIT2 (FIGs. 4E- 4F), which is notable as the ROBO1-SLIT2 axis has been shown to promote metastasis in pancreatic cancer (Li et al., 2023). While ROBO-SLIT complexes are annotated as potential integrin interactors at the pathway level, no direct interactions at the PPI level had been previously documented (Wu et al., 2017). Additionally, BM1 and 231 cells had both shared and unique ephrin receptors, which are receptor tyrosine kinases strongly implicated in cancer (FIGs. 4D, 4F) (Pasquale et al., 2024). For example, EPHA2 and EPHB2 were proximal to integrins at roughly similar levels in 231 and BM1 cells, but other ephrins showed differential proximity across cell types. EPHB4 integrin proximity was increased in 231 cells, whereas EPHA4 and EPHB3 integrin proximity was uniquely detected in post-metastatic BM1 cells (FIGs. 4D, 4F), matching their previously identified roles in promoting cancer progression (Lu et al., 2014; Furuhashi et al., 2021; Ji et al., 2011; Maddigan et al., 2011). These data presented the potential for quantitative and comparative RGDL-PhotoPPI profiling to identify ‘AND- gate’ complex pairs (i.e., the proximity of integrins and identified surface markers on live cells, FIG. 4G) that could serve as novel players in breast cancer metastasis and post-metastatic colonization, as well as potential markers for diagnostic or therapeutic interventions.

[0285] To evaluate whether the unique integrin social network members might serve as prognostic markers, the inventors assembled a 20-protein signature, or ‘interaction barcode,’ for BM1 (metastatic) and MDA-MB-231 cells (primary) by selecting the most abundant and unique surface proteins within the enriched RGDL-labeled integrin social network in each cell line (FIG. 9E). Since broad-scale clinical databases with matched protein interaction partner information do not exist, the inventors reasoned that the mRNA expression of these targets within annotated breast cancer patient databases could serve as a pragmatic - albeit imperfect - proxy. Therefore, the inventors performed retrospective survival analyses of breast cancerpatients by comparing relative mRNA expression levels of each interaction partner signature (FIG. 5A) within published databases of TNBC and breast cancer patient populations (Gyorffy et al., 2021; Nagy et al., 2021) (FIGs. 5A-5B). Surprisingly, the inventors found that TNBC patients with higher mRNA expression levels of the primary tumor barcode (e.g., 231 cell- derived integrin partners) had a significant survival advantage as compared to patients with lower expression levels (FIG. 5A; Hazard Ratio, HR = 0.5, p = 0.008). Expression of the metastatic integrin interactor barcode (e.g., those unique to BM1 cells) was significantly correlated with increased mortality in TNBC patients (HR = 1.92, / ? = 0.01, FIG. 5B). These trends held true even amongst a larger cohort of 1,089 patients with diverse forms of breast cancer (HR = 0.51 and HR = 1.63 for the primary and metastatic barcodes, respectively; FIG. 5B), thus unveiling significant correlations between integrin social networks of metastatic cells — identified through unbiased RGDL-PhotoPPI profiling — and patient outcomes. Collectively, these data may offer new avenues for targeted therapy and prognostic marker development in aggressive breast cancer types.6. Discussion

[0286] In this Example, the inventors aimed to develop a photoproximity profiling method capable of interrogating integrin-centered protein interactors, or ‘social networks,’ directly on live cells. To do so, the inventors developed an integrin sub-family targeting RGDL probe that could localize to and proximally activate a reactive phenoxyl radical tagging species in response to light. The inventors demonstrated several advantages to this approach. First, by bypassing the use of genetic fusion proteins and / or antibody-based recognition, RGDL- PhotoPPI could probe heterogeneous, native integrin complexes that occurred on live cells and tissues. In situ labeled protein retrieval, detection, and quantification by MS-based proteomics enabled unbiased discovery of interacting target proteins without a priori information about their identities. Moreover, this approach allowed for qualitative and quantitative comparison of integrin social networks between diverse tumor cell types, microenvironmental contexts, and metastatic status. Studies of surface complex dynamics and the impact of microenvironmental conditions, in particular, can be sensitive to external stimuli, and therefore the use of rapid light activation as a proximity trigger is minimally perturbative relative to other proximity profiling approaches. The speed of RGDL-PhotoPPI photoactivation was also noteworthy, demonstrated by robust proteome coverage and high enrichment over background following irradiation times as low as one minute. Future studies could leverage this temporalresolution to detect dynamic integrin interactors across their lifecycle and in different sub- cellular locales following defined perturbations.

[0287] While it is commonly understood that integrin signaling plays a role in basal cellular function, the results found by the inventors revealed significant differences in integrin interactomes across different cancer cell lines and contexts. RGDL-PhotoPPI detected numerous previously validated integrin interactors, including cell surface growth factor receptors like EGFR, ERBB2 and MET, as well as known and novel proteins involved in ECM remodeling, focal adhesion complexes and extracellular proteases (FIG. 2). Within these larger protein groups, the inventors identified considerable differences between cell types. For instance, integrin networks were enriched for endosomal and focal adhesion complex interactors in epithelial cervical cancer (HeLa) cells relative to the invasive glioblastoma (U87- MG) cells, which showed higher enrichment for interaction partners implicated in proliferative signaling, migration, and ECM remodeling. Intriguingly, the inventors detected significant rewiring of integrin-centered social networks when comparing adherent versus detached cell populations from HeLa and U87-MG tumor cells. This result was not entirely surprising, given the role of integrins in ECM interactions and migration, however it raises the possibility of mapping and perhaps targeting distinct extracellular complexes that differ based on tumor cell microenvironments. Coupled with the ability to directly profile fresh tumor samples, the inventors posited that RGDL-PhotoPPI could be applied in the future to map cell surface migratory hubs more broadly across normal and diseased tissue samples.

[0288] The sensitive and quantitative detection of integrin social networks is perhaps most useful to identify ‘AND-gate’ protein complexes that differentiate cells of different disease potential, such as the pre- and post-metastatic TNBC cell lines. While the comparison of 231 and BM1 cells confirmed that many integrin targets and interactors were similar, the inventors detected many novel interactors previously associated with cancer progression and metastasis that were uniquely present in one cell line. The detected Ephrin and NRP / ROBO subcomplexes that were present in either pre- or post-metastatic TNBC cells provided potential hypotheses connecting cell migratory and signaling complexes co-localizing and perhaps forming synergistic ‘signalosomes.’ Further investigation is needed to elucidate the roles of these newly identified interactions in cancer progression and metastasis.Finally, the inventors used photoproximity profiling for the first time to identify metastasisassociation interaction ‘barcode’ protein sets (FIG. 5C). Intriguingly, the application of these social networks to clinical databases uncovered significant survival advantages for patients with higher expression of the pre-metastatic interactors (derived from MDA-MB-231 cells)and significantly reduced survival among patients with higher expression of post-metastatic interactors (derived from BM1 cells). These results raised the intriguing possibility that combinations of receptors, even when anchored around seemingly ubiquitous surface proteins like integrins, could serve as markers of and potential targets for tumor metastasis or other disease states. For example, the identification of select integrin-proximity of proteins like VCAM1, which has been targeted in breast cancer, in 231 vs. BM1 cells could suggest differential therapeutic efficacy based on breast cancer stage and support diagnostic profiling by RGDL-PhotoPPI as a method to confirm therapeutic potential for certain patients or cancer sub-types. Coupled with the ability to directly profile interactors on fresh tissues, this raises the possibility of RGDL-PhotoPPI profiling of patient samples for further discovery of select interaction markers with potential diagnostic utility. A caveat with and limitation of these data was that clinical databases of integrin-centered or other surface receptor-centered interactions were not available for review, requiring mRNA levels to be queried here as a proxy. Taken together, the inventors posited that further development of proximity profiling methods like RGDL-PhotoPPI will provide opportunities to draft high-quality, spatially-resolved maps of integrin and other protein interaction social networks directly in physiologically and diseaserelevant contexts.* * *

[0289] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A photo-dependent proximity labeling system comprising: an activatable probe comprising a ligand covalently attached to a photosensitizer molecule; and a retrievable substrate, wherein the activatable probe and the retrievable substrate are different molecules.

2. The photo-dependent proximity labeling system of claim 1, wherein the ligand comprises an Arg-Gly-Asp peptide, or a functional derivative thereof.

3. The photo-dependent proximity labeling system of claim 1, wherein the ligand consists of an Arg-Gly-Asp peptide, or a functional derivative thereof.

4. The photo-dependent proximity labeling system of claim 1, wherein the ligand comprises a peptide, small molecule, or biologic.

5. The photo-dependent proximity labeling system of any one of claims 1-4, wherein the photosensitizer molecule comprises lumichrome, or a functional derivative thereof.

6. The photo-dependent proximity labeling system of any one of claims 1-5, wherein the retrievable substrate comprises a structure of BP, AP, SI, S2, S3 or S4:

7. The photo-dependent proximity labeling system of any one of claims 1-6, wherein the retrievable substrate comprises a biotin linked to a phenol derivative, a diazirine derivative, a benzophenone derivative, an amine derivative, or an aryl azide derivative.

8. The photo-dependent proximity labeling system of any one of claims 1-7, wherein the activatable probe comprises a molecule having a structure of Formula (I):Formula I9. The photo-dependent proximity labeling system of any one of claims 1-8, wherein the ligand is an integrin ligand.

10. The photo-dependent proximity labeling system of any one of claims 1-9, wherein the photoactivatable probe is activatable by light of a wavelength of 440 and / or 365 nm.

11. A photo-dependent proximity labeling system comprising a retrievable substrate; and a molecule having a structure of Formula (I):Formula I12. The photo-dependent proximity labeling system of claim 11, wherein the retrievable substrate comprises a structure of formula BP:Biotin Phenol (BP)13. A method of labeling a protein complex in a sample, the method comprising: introducing the photo-dependent proximity labeling system of any one of claims 1-12 to the sample; and exposing the sample to a wavelength of light capable of activating the activatable probe.

14. The method of claim 13, wherein introducing the photo-dependent proximity labeling system comprises introducing an activatable probe and a retrievable substrate prior to exposing the sample to light.

15. The method of claim 13 or 14, wherein introducing the photo-dependent proximity labeling system comprises the sequential steps of: introducing an activatable probe, introducing a retrievable substrate, and exposing the sample to light.

16. The method of any one of claims 13-15, wherein the protein complex comprises an integrin.

17. The method of any one of claims 13-16, wherein the sample comprises a population of cells.

18. The method of any one of claims 13-17, wherein the sample is a biological sample from a patient.

19. The method of any one of claims 13-18, wherein the biological sample comprises a fresh sample, a frozen sample, and / or a fixed sample.

20. The method of any one of claims 13-19, wherein the biological sample is not frozen and / or not fixed.

21. The method of any one of claims 13-20, wherein the sample is a biopsy sample.

22. The method of any one of claims 13-21, wherein the sample is from an individual having, suspected of having, or diagnosed with having cancer.

23. The method of any one of claims 13-22, wherein the photo-dependent proximity labeling system is introduced at a concentration of at least, at most, approximately, or exactly 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441,442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477,478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495,496, 497, 498, 499, 500 pM, or any range derivable therein.

24. The method of any one of claims 13-23, wherein the wavelength of light is 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 nm, or any range derivable therein.

25. The method of any one of claims 13-24, wherein the exposing occurs for between approximately 1 second to 5 minutes.

26. The method of any one of claims 13-25, further comprising retrieving proteins labeled with the retrievable substrate from the sample.

27. The method of claim 26, wherein the retrieving comprises streptavidin enrichment.

28. The method of any one of claims 13-27, further comprising analyzing the sample to determine an identity of proteins labeled with the retrievable substrate.

29. The method of claim 28, wherein the analyzing comprises performing mass spectrometry, chromatography, electrophoresis, and / or a suitable protein identification assay.

30. A method of labeling a protein complex in a sample, the method comprising the steps of: introducing a retrievable substrate comprising biotin and a molecule having a structure of Formula (I) to the sample; exposing the sample to a wavelength of light capable of activating the structure of Formula (I) thereby generating a set of biotin labeled proteins; retrieving the set of labeled proteins with a streptavidin enrichment to generate a set of retrieved proteins; and analyzing the set of retrieved proteins, wherein Formula (I) is:Formula I31. The method of claim 30, wherein the sample is from an individual having, suspected of having, having symptoms of, or diagnosed with having cancer.

32. A method of identifying one or more therapeutic targets in a patient, the method comprising: introducing the photo-dependent proximity labeling system of any one of claims 1-12 to a biological sample from the patient, wherein the activatable probe binds to a protein of interest; and exposing the sample to a wavelength of light capable of activating the activatable probe to generate a set of labeled proteins.

33. The method of claim 32, wherein introducing the photo-dependent proximity labeling system comprises introducing an activatable probe and a retrievable substrate prior to exposing the sample to light.

34. The method of claim 32 or 33, wherein introducing the photo-dependent proximity labeling system comprises the sequential steps of: introducing an activatable probe, introducing a retrievable substrate, and exposing the sample to light.

35. The method of any one of claims 32-34, wherein the set of labeled proteins comprises at least one integrin.

36. The method of any one of claims 32-35, wherein the biological sample is a biopsy sample.

37. The method of any one of claims 32-36, wherein the biological sample comprises a fresh sample, a frozen sample, and / or a fixed sample.

38. The method of any one of claims 32-37, wherein the biological sample is not frozen and / or not fixed.

39. The method of any one of claims 32-38, wherein the photo-dependent proximity labeling system is introduced at a concentration of at least, at most, approximately, or exactly 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 29, 30, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441,442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477,478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495,496, 497, 498, 499, 500 pM, or any range derivable therein.

40. The method of any one of claims 32-39, wherein the wavelength of light is 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392,393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 nm, or any range derivable therein.

41. The method of any one of claims 32-40, wherein the exposing occurs for between approximately 1 second to 5 minutes.

42. The method of any one of claims 32-41, wherein the patient has, is suspected of having, or has been diagnosed with having cancer.

43. The method of any one of claims 32-42, further comprising retrieving proteins labeled with the retrievable substrate from the sample.

44. The method of claim 43, wherein the retrieving comprises streptavidin enrichment.

45. The method of any one of claims 32-44, further comprising analyzing the sample to determine an identity of proteins labeled with the retrievable substrate.

46. The method of claim 45, wherein the analyzing comprises performing mass spectrometry, chromatography, electrophoresis, and / or a suitable protein identification assay.

47. The method of any one of claims 32-46, wherein the therapeutic targets are proximal to the protein of interest in the sample.

48. The method of any one of claims 32-47, wherein the therapeutic targets are within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461,462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497,498, 499, 500 angstroms of the protein of interest in the sample.

49. The method of any one of claims 32-48, wherein the protein of interest comprises an integrin.

50. The method of any one of claims 32-49, wherein the protein of interest comprises an oncogenic protein.

51. An engineered activatable probe comprising a ligand covalently attached to a photosensitizer molecule, wherein the ligand comprises a peptide, small molecule, or biologic; and the photosensitizer molecule comprises lumichrome or a functional derivative thereof.

52. The engineered activatable probe of claim 51, wherein the activatable probe comprises a molecule having a structure of Formula (I):Formula I53. A method of diagnosing cancer in a patient, said method comprising: introducing the photo-dependent proximity labeling system of any one of claims 1-12 to a biological sample obtained from the patient, wherein the activatable probe binds to a protein of interest; exposing the sample to a wavelength of light capable of activating the activatable probe to generate a set of labeled proteins; and determining the identity of one or more proteins in the set of labeled proteins.

54. The method of claim 53, wherein introducing the photo-dependent proximity labeling system comprises introducing an activatable probe and a retrievable substrate prior to exposing the sample to light.

55. The method of claim 53 or 54, wherein introducing the photo-dependent proximity labeling system comprises the sequential steps of: introducing an activatable probe, introducing a retrievable substrate, and exposing the sample to light.

56. The method of any one of claims 53-55, wherein the patient is a human patient.

57. The method of any one of claims 53-56, wherein the biological sample comprises tissue from a cancer.

58. The method of any one of claims 53-57, wherein the labeled proteins are enriched at least about 2-fold.

59. The method of any one of claims 53-58, wherein the protein of interest is an integrin.

60. The method of any one of claims 53-59, wherein the labeled proteins comprise ITGB4- isoform 2, CD55, MELTF, NECTIN2, DLG1, PTPRK, CSF2RA, FRMD6, VASN, FCGRT, PCDHB13, CA12, HRH1, SLC3A2-1, VCAM-1, EPHB3, EPHA4, SLIT2, FAP,CD44, TGFB1, CTNNB1, PCDHGC3, BTN3A3, BCAP31, CD74, NRP1, ERBB2, EPHB2, EPHB4, EPHA2, PLXND1, TGFBR2, NRP2, SLC3A2-2, ITGA6, CD55-isoform 5, HK2, NECTIN2-isoform 2, R0B01, SLC12A4-isoform 7, TENM1, DYSF, ATP11C, SEMA6B, OCLN, ICAM2, NRG1, SNAP23, RELL1, SDC1, SLC16A7, EMP3, NPTN, or a combination thereof.6E The method of any one of claims 53-60, further comprising administering an effective amount of a treatment to the patient.

62. The method of claim 61, wherein the treatment comprises an inhibitor of ITGB4-isoform 2, CD55, MELTF, NECTIN2, DLG1, PTPRK, CSF2RA, FRMD6, VASN, FCGRT, PCDHB13, CA12, HRH1, SLC3A2-1, VCAM-1, EPHB3, EPHA4, SLIT2, FAP, CD44, TGFB1, CTNNB1, PCDHGC3, BTN3A3, BCAP31, CD74, NRP1, ERBB2, EPHB2, EPHB4, EPHA2, PLXND1, TGFBR2, NRP2, SLC3A2-2, ITGA6, CD55-isoform 5, HK2, NECTIN2-isoform 2, ROBO1, SLC12A4-isoform 7, TENM1, DYSF, ATP11C, SEMA6B, OCLN, ICAM2, NRG1, SNAP23, RELL1, SDC1, SLC16A7, EMP3, NPTN, or a combination thereof.

63. The method of claim 61 or 62, wherein the treatment is an anti-metastatic treatment.

64. A method of identifying binding partners of a protein of interest in a biological sample obtained from a patient, said method comprising: introducing the photo-dependent proximity labeling system of any one of claims 1-12 to the biological sample wherein the activatable probe binds to the protein of interest; exposing the sample to a wavelength of light capable of activating the activatable probe to generate a set of labeled proteins.

65. The method of claim 64, wherein introducing the photo-dependent proximity labeling system comprises introducing an activatable probe and a retrievable substrate prior to exposing the sample to light.

66. The method of claim 64 or 65, wherein introducing the photo-dependent proximity labeling system comprises the sequential steps of: introducing an activatable probe, introducing a retrievable substrate, and exposing the sample to light.

67. The method of any one of claims 64-66, wherein the set of labeled proteins are identified as proteins that bind to the protein of interest.

68. The method of any one of claims 64-67, wherein the biological sample obtained from a patient is obtained from a human patient.

69. The method of any one of claims 64-68, wherein the biological sample obtained from a patient comprises tissue from a cancer.

70. The method of any one of claims 64-69, wherein the binding partners are enriched at least about 2-fold.

71. The method of any one of claims 64-70, wherein the protein of interest is an integrin.

72. The method of any one of claims 64-71, wherein the labeled proteins comprise ITGB4- isoform 2, CD55, MELTF, NECTIN2, DLG1, PTPRK, CSF2RA, FRMD6, VASN, FCGRT, PCDHB13, CA12, HRH1, SLC3A2-1, VCAM-1, EPHB3, EPHA4, SLIT2, FAP, CD44, TGFB1, CTNNB1, PCDHGC3, BTN3A3, BCAP31, CD74, NRP1, ERBB2, EPHB2, EPHB4, EPHA2, PLXND1, TGFBR2, NRP2, SLC3A2-2, ITGA6, CD55-isoform 5, HK2, NECTIN2-isoform 2, ROBO1, SLC12A4-isoform 7, TENM1, DYSF, ATP11C, SEMA6B, OCLN, ICAM2, NRG1, SNAP23, RELL1, SDC1, SLC16A7, EMP3, NPTN, or a combination thereof.

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