活体动物中酪氨酸磷酸化及其介导的蛋白质复合物研究

酪氨酸磷酸化（Tyrosine phosphorylation, pTyr）是一种常见的蛋白 质翻译后修饰过程，广泛参与机体免疫调节、肿瘤发生发展等重要生命活 动。其丰度较低，难以被质谱直接检测。蛋白质特定位点的酪氨酸发生磷 酸化后，能够在分秒尺度招募相关蛋白质形成复合物，从而发挥功能。为 了全面构建 pTyr 介导的信号转导网络，传统方法常使用永生化的细胞系， 利用pTyr 抗体富集以及亲和纯化质谱联用技术分别实现位点和复合物的检 测。但该策略丧失了组织层面的时空特异性信息，容易丢失弱的和瞬时的 相互作用，所构建的信号网络与真实生理条件下差异较大。 为解决上述问题，本研究首先基于SH2（Src homology 2, SH2）超亲 体和固定化钛离子亲和色谱法联用的高效富集策略，建立了一套用于原代 细胞及活体动物的 pTyr 位点分析方法。利用该方法针对脾脏分离培养获得 的原代T细胞，解析了抗体激活条件下的282个关键pTyr位点信息，并比 较了与永生化细胞系所获得的数据之间的差异。进一步，将该方法拓展应 用至活体动物组织层面，较为全面地解析了肝脏组织在表皮生长因子 （Epidermal growth factor, EGF）刺激条件下的7 个不同时间点的 1027个 位点信息，初步绘制了表皮生长因子受体（Epidermal growth factor receptor, EGFR）信号通路分钟分辨率的 pTyr位点时序性变化图谱。 在此基础上，结合活体动物近程标记方法，对 pTyr介导的动态蛋白质 复合物进行同步鉴定。在活体小鼠肝脏原位解析了EGF刺激2 min条件下 显著上调的 85个位点信息以及54个与生长因子受体结合蛋白 2相互作用 的动态蛋白质复合物，最终实现了位点和复合物二维信息的采集。进一 步，将该研究策略应用至肝脏再生小鼠模型，解析了肝脏再生过程中关键 的pTyr 位点变化以及潜在形成的动态蛋白质复合物。 综上所述，本研究建立了一套能够同步采集 pTyr位点和动态蛋白质复 合物信息的二维分析流程，为解析原代细胞乃至活体动物组织层面更真实 的pTyr 信号转导网络奠定了方法学基础。

[1] BLUDAU I, AEBERSOLD R. Proteomic and interactomic insights into the molecular basis of cell functional diversity[J]. Nat. Rev. Mol. Cell Biol., 2020, 21(6): 327-340.
[2] ROZAKIS-ADCOCK M, FERNLEY R, WADE J, et al. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1[J]. Nature, 1993, 363(6424): 83-85.
[3] OLIVIER J P, RAABE T, HENKEMEYER M, et al. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos[J]. Cell, 1993, 73(1): 179-191.
[4] WAKSMAN G, KOMINOS D, ROBERTSON S C, et al. Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides[J]. Nature, 1992, 358(6388): 646-653.
[5] SONGYANG Z, CANTLEY L C. ZIP codes for delivering SH2 domains[J]. Cell, 2004, 116(2 Suppl): S41-S43.
[6] WU P, NIELSEN T E, CLAUSEN M H. Small-molecule kinase inhibitors: an analysis of FDA-approved drugs[J]. Drug Discovery Today, 2016, 21(1): 5-10.
[7] ATTWOOD M M, FABBRO D, SOKOLOV A V, et al. Trends in kinase drug discovery: targets, indications and inhibitor design[J]. Nat. Rev. Drug Discovery, 2021, 20(11): 839-861.
[8] HEIL L R, DAMOC E, ARREY T N, et al. Evaluating the performance of the astral mass analyzer for quantitative proteomics using data-independent acquisition[J]. J. Proteome Res., 2023, 22(10): 3290-3300.
[9] CHEN W, CHEN L, TIAN R. An integrated strategy for highly sensitive phosphoproteome analysis from low micrograms of protein samples[J]. Analyst, 2018, 143(15): 3693-3701.
[10] MAO Y, CHEN P, KE M, et al. Fully integrated and multiplexed sample preparation technology for sensitive interactome profiling[J]. Anal. Chem., 2021, 93(5): 3026-3034.
[11] CHEN W, WANG S, ADHIKARI S, et al. Simple and integrated spintip-based technology applied for deep proteome profiling[J]. Anal. Chem., 2016, 88(9): 4864-4871.
[12] YAO Y, WANG Y, WANG S, et al. One-step SH2 superbinder-based approach for sensitive analysis of tyrosine phosphoproteome[J]. J. Proteome Res., 2019, 18(4): 1870-1879.
[13] AHSAN N, WILSON R S, RAO R S P, et al. Mass spectrometry-based identification of phospho-Tyr in plant proteomics[J]. J. Proteome Res., 2020, 19(2): 561-571.
[14] URBAN J. A review on recent trends in the phosphoproteomics workflow. From sample preparation to data analysis[J]. Anal. Chim. Acta., 2022, 1199: 338857-338877.
[15] BIAN Y, LI L, DONG M, et al. Ultra-deep tyrosine phosphoproteomics enabled by a phosphotyrosine superbinder[J]. Nat. Chem. Biol., 2016, 12(11): 959-966.
[16] BISSON N, JAMES D A, IVOSEV G, et al. Selected reaction monitoring mass spectrometry reveals the dynamics of signaling through the GRB2 adaptor[J]. Nat. Biotechnol., 2011, 29(7): 653-658.
[17] KE M, LIU J, CHEN W, et al. Integrated and quantitative proteomic approach for charting temporal and endogenous protein complexes[J]. Anal. Chem., 2018, 90(21): 12574-12583.
[18] LIU J, YANG L, HE A, et al. Stable and EGF-induced temporal interactome profiling of CBL and CBLB highlights their signaling complex diversity[J]. J. Proteome Res., 2021, 20(7): 3709-3719.
[19] KE M, YUAN X, HE A, et al. Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics[J]. Nat. Commun., 2021, 12(1): 71.
[20] QIN W, CHO K F, CAVANAGH P E, et al. Deciphering molecular interactions by proximity labeling[J]. Nat. Methods, 2021, 18(2): 133-143.
[21] LAM S S, MARTELL J D, KAMER K J, et al. Directed evolution of APEX2 for electron microscopy and proximity labeling[J]. Nat. Methods, 2015, 12(1): 51-54.
[22] KONG Q, KE M, WENG Y, et al. Dynamic phosphotyrosine-dependent signaling profiling in living cells by two-dimensional proximity proteomics[J]. J. Proteome Res., 2022, 21(11): 2727-2735.
[23] ABRAHAM R T, WEISS A. Jurkat T cells and development of the T-cell receptor signalling paradigm[J]. Nat. Rev. Immunol., 2004, 4(4): 301-308.
[24] ASTOUL E, EDMUNDS C, CANTRELL D A, et al. PI 3-K and T-cell activation: limitations of T-leukemic cell lines as signaling models[J]. Trends Immunol., 2001, 22(9): 490-496.
[25] CARON E, RONCAGALLI R, HASE T, et al. Precise temporal profiling of signaling complexes in primary cells using SWATH mass spectrometry[J]. Cell Rep., 2017, 18(13): 3219-3226.
[26] YU J S. From discovery of tyrosine phosphorylation to targeted cancer therapies: The 2018 Tang Prize in Biopharmaceutical Science[J]. Biomed. J., 2019, 42(2): 80-83.
[27] SHI Y, GAO W, LYTLE N K, et al. Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring[J]. Nature, 2019, 569(7754): 131-135.
[28] TREASE A J, LI H, SPAGNOL G, et al. Regulation of Connexin32 by ephrin receptors and T-cell protein-tyrosine phosphatase[J]. J. Biol. Chem., 2019, 294(1): 341-350.
[29] BOROWICZ P, SUNDVOLD V, CHAN H, et al. Tyr(192) regulates lymphocyte-specific tyrosine kinase activity in T cells[J]. J. Immunol., 2021, 207(4): 1128-1137.
[30] TERADA Y, HIGASHI N, HIDAKA Y, et al. Protein tyrosine phosphatase inhibitor, orthovanadate, induces contraction via Rho kinase activation in mouse thoracic aortas[J]. Biol. Pharm. Bull., 2019, 42(6): 877-885.
[31] LI J, ZHAN X. Mass spectrometry analysis of phosphotyrosine-containing proteins[J]. Mass Spectrom. Rev., 2023,1-31.
[32] HUNTER T. The genesis of tyrosine phosphorylation[J]. Cold Spring Harbor Perspect. Biol., 2014, 6(5): 20644-20658.
[33] BELTRAN L, CUTILLAS P R. Advances in phosphopeptide enrichment techniques for phosphoproteomics[J]. Amino Acids, 2012, 43(3): 1009-1024.
[34] GEMBITSKY D S, LAWLOR K, JACOVINA A, et al. A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation[J]. Mol. Cell. Proteomics, 2004, 3(11): 1102-1118.
[35] TINTI M, NARDOZZA A P, FERRARI E, et al. The 4G10, pY20 and p-Tyr-100 antibody specificity: profiling by peptide microarrays[J]. Nat. Biotechnol., 2012, 29(5): 571-577.
[36] STANFORD S M, BOTTINI N. Targeting protein phosphatases in cancer immunotherapy and autoimmune disorders[J]. Nat. Rev. Drug Discovery, 2023, 22(4): 273-294.
[37] DIOP A, SANTORELLI D, MALAGRINò F, et al. SH2 Domains: folding, binding and therapeutical approaches[J]. Int. J. Mol. Sci., 2022, 23(24):15944-15967.
[38] HORNBECK P V, CHABRA I, KORNHAUSER J M, et al. PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation[J]. Proteomics, 2004, 4(6): 1551-1561.
[39] KANEKO T, HUANG H, CAO X, et al. Superbinder SH2 domains act as antagonists of cell signaling[J]. Sci. Signal., 2012, 5(243): 68-78.
[40] LADBURY J E, AROLD S T. Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling[J]. Methods Enzymol., 2011, 488: 147-183.
[41] JONES R B, GORDUS A, KRALL J A, et al. A quantitative protein interaction network for the ErbB receptors using protein microarrays[J]. Nature, 2006, 439(7073): 168-174.
[42] LADBURY J E, LEMMON M A, ZHOU M, et al. Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal[J]. Proc. Natl. Acad. Sci. U. S. A., 1995, 92(8): 3199-3203.
[43] DONG M, BIAN Y, WANG Y, et al. Sensitive, robust, and cost-effective approach for tyrosine phosphoproteome analysis[J]. Anal. Chem., 2017, 89(17): 9307-9314.
[44] KONG Q, WENG Y, ZHENG Z, et al. Integrated and high-throughput approach for sensitive analysis of tyrosine phosphoproteome[J]. Anal. Chem., 2022, 94(40): 13728-13736.
[45] LUNDBY A, FRANCIOSA G, EMDAL K B, et al. Oncogenic mutations rewire signaling pathways by switching protein recruitment to phosphotyrosine sites[J]. Cell, 2019, 179(2): 543-560.
[46] HUTTLIN E L, JEDRYCHOWSKI M P, ELIAS J E, et al. A tissue-specific atlas of mouse protein phosphorylation and expression[J]. Cell, 2010, 143(7): 1174-1189.
[47] DITTMANN A, KENNEDY N J, SOLTERO N L, et al. High-fat diet in a mouse insulin-resistant model induces widespread rewiring of the phosphotyrosine signaling network[J]. Mol. Syst. Biol., 2019, 15(8): 8849-8871.
[48] TIAN R, WANG H, GISH G D, et al. Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor[J]. Proc. Natl. Acad. Sci. U. S. A., 2015, 112(13): 1594-1603.
[49] CONDE J N. Yeast two-hybrid system for mapping novel dengue protein interactions[J]. Methods Mol. Biol., 2022, 2409: 119-132.
[50] FIELDS S, STERNGLANZ R. The two-hybrid system: an assay for protein-protein interactions[J]. Trends Genet., 1994, 10(8): 286-292.
[51] LUCK K, KIM D K, LAMBOURNE L, et al. A reference map of the human binary protein interactome[J]. Nature, 2020, 580(7803): 402-408.
[52] YOON T Y, LEE H W. Shedding light on complexity of protein-protein interactions in cancer[J]. Curr. Opin. Chem. Biol., 2019, 53: 75-81.
[53] ZHENG J, CHEN X, YANG Y, et al. Mass spectrometry-based protein complex profiling in time and space[J]. Anal. Chem., 2021, 93(1): 598-619.
[54] HAN S, LI J, TING A Y. Proximity labeling: spatially resolved proteomic mapping for neurobiology[J]. Curr. Opin. Neurobiol., 2018, 50: 17-23.
[55] GINGRAS A C, ABE K T, RAUGHT B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles[J]. Curr. Opin. Chem. Biol., 2019, 48: 44-54.
[56] KUSHNER J, PAPA A, MARX S O. Use of proximity labeling in cardiovascular research[J]. JACC Basic Transl. Sci., 2021, 6(7): 598-609.
[57] CHOI-RHEE E, SCHULMAN H, CRONAN J E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase[J]. Protein Sci., 2004, 13(11): 3043-3050.
[58] ROUX K J, KIM D I, RAIDA M, et al. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells[J]. J. Cell Biol., 2012, 196(6): 801-810.
[59] BRANON T C, BOSCH J A, SANCHEZ A D, et al. Efficient proximity labeling in living cells and organisms with TurboID[J]. Nat. Biotechnol., 2018, 36(9): 880-887.
[60] LEE S Y, CHEAH J S, ZHAO B, et al. Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells[J]. Nat. Methods, 2023, 20(6): 908-917.
[61] MARTELL J D, DEERINCK T J, SANCAK Y, et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy[J]. Nat. Biotechnol., 2012, 30(11): 1143-1148.
[62] LOH K H, STAWSKI P S, DRAYCOTT A S, et al. Proteomic analysis of unbounded cellular compartments: synaptic clefts[J]. Cell, 2016, 166(5): 1295-1307.
[63] LI J, HAN S, LI H, et al. Cell-surface proteomic profiling in the fly brain uncovers wiring regulators[J]. Cell, 2020, 180(2): 373-386.
[64] XIE Q, LI J, LI H, et al. Transcription factor Acj6 controls dendrite targeting via a combinatorial cell-surface code[J]. Neuron, 2022, 110(14): 2299-2314.
[65] SHUSTER S A, LI J, CHON U, et al. In situ cell-type-specific cell-surface proteomic profiling in mice[J]. Neuron, 2022, 110(23): 3882-3896.
[66] MANDELMAN D, SCHWARZ F P, LI H, et al. The role of quaternary interactions on the stability and activity of ascorbate peroxidase[J]. Protein Sci., 1998, 7(10): 2089-2098.
[67] RHEE H W, ZOU P, UDESHI N D, et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging[J]. Science, 2013, 339(6125): 1328-1331.
[68] PAEK J, KALOCSAY M, STAUS D P, et al. Multidimensional tracking of GPCR signaling via peroxidase-catalyzed proximity labeling[J]. Cell, 2017, 169(2): 338-349.
[69] LOBINGIER B T, HüTTENHAIN R, EICHEL K, et al. An approach to spatiotemporally resolve protein interaction networks in living cells[J]. Cell, 2017, 169(2): 350-360.
[70] PEREZ VERDAGUER M, ZHANG T, SURVE S, et al. Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR[J]. Cell Rep., 2022, 39(11): 110950-110987.
[71] KE M, YUAN X, HE A, et al. Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics[J]. Nat. Commun., 2021, 12(1): 71-84.
[72] ZHOU Y, ZOU P. The evolving capabilities of enzyme-mediated proximity labeling[J]. Curr. Opin. Chem. Biol., 2021, 60: 30-38.
[73] UEZU A, KANAK D J, BRADSHAW T W, et al. Identification of an elaborate complex mediating postsynaptic inhibition[J]. Science, 2016, 353(6304): 1123-1129.
[74] DINGAR D, KALKAT M, CHAN P K, et al. BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors[J]. J. Proteomics, 2015, 118: 95-111.
[75] DROUJININE I A, MEYER A S, WANG D, et al. Proteomics of protein trafficking by in vivo tissue-specific labeling[J]. Nat. Commun., 2021, 12(1): 2382-2404.
[76] KIM K E, PARK I, KIM J, et al. Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice[J]. Nat. Commun., 2021, 12(1): 5204-5213.
[77] WEI W, RILEY N M, YANG A C, et al. Cell type-selective secretome profiling in vivo[J]. Nat. Chem. Biol., 2021, 17(3): 326-334.
[78] KLEIMAN L B, MAIWALD T, CONZELMANN H, et al. Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding[J]. Mol. Cell, 2011, 43(5): 723-737.
[79] ARDITO F, GIULIANI M, PERRONE D, et al. The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy[J]. Int. J. Mol. Med., 2017, 40(2): 271-280.
[80] SABLINA A A, BUDANOV A V, ILYINSKAYA G V, et al. The antioxidant function of the p53 tumor suppressor[J]. Nat. Med., 2005, 11(12): 1306-1313.
[81] NAKAMURA J, PURVIS E R, SWENBERG J A. Micromolar concentrations of hydrogen peroxide induce oxidative DNA lesions more efficiently than millimolar concentrations in mammalian cells[J]. Nucleic Acids Res., 2003, 31(6): 1790-1795.
[82] DUMRONGPRECHACHAN V, SALISBURY R B, SOTO G, et al. Cell-type and subcellular compartment-specific APEX2 proximity labeling reveals activity-dependent nuclear proteome dynamics in the striatum[J]. Nat. Commun., 2021, 12(1): 4855-4871.
[83] LIU G, PAPA A, KATCHMAN A N, et al. Mechanism of adrenergic Ca(V)1.2 stimulation revealed by proximity proteomics[J]. Nature, 2020, 577(7792): 695-700.
[84] DONG D, ZHENG L, LIN J, et al. Structural basis of assembly of the human T cell receptor-CD3 complex[J]. Nature, 2019, 573(7775): 546-552.
[85] WANG W, AI X. Primary culture of immature, naïve mouse CD4(+) T cells[J]. STAR Protoc., 2021, 2(3): 100756-100778.
[86] POLONI C, SCHONHOFER C, IVISON S, et al. T-cell activation-induced marker assays in health and disease[J]. Immunol. Cell Biol., 2023, 101(6): 491-503.
[87] TAN X, QI C, ZHAO X, et al. ERK inhibition promotes engraftment of allografts by reprogramming T-Cell metabolism[J]. Adv. Sci. (Weinh), 2023, 10(16): 2206768-2206786.
[88] KIM J E, WHITE F M. Quantitative analysis of phosphotyrosine signaling networks triggered by CD3 and CD28 costimulation in Jurkat cells[J]. Ja. Immunol., 2006, 176(5): 2833-2843.
[89] TAKEMOTO Y, SATO M, FURUTA M, et al. Distinct binding patterns of HS1 to the Src SH2 and SH3 domains reflect possible mechanisms of recruitment and activation of downstream molecules[J]. Int. Immunol., 1996, 8(11): 1699-1705.
[90] RIECKMANN J C, GEIGER R, HORNBURG D, et al. Social network architecture of human immune cells unveiled by quantitative proteomics[J]. Nat. Immunol., 2017, 18(5): 583-593.
[91] WANGE R L, GUITIáN R, ISAKOV N, et al. Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70[J]. J. Biol. Chem., 1995, 270(32): 18730-18733.
[92] WATTS J D, AFFOLTER M, KREBS D L, et al. Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70[J]. J. Biol. Chem., 1994, 269(47): 29520-29529.
[93] HUMPHREY S J, AZIMIFAR S B, MANN M. High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics[J]. Nat. Biotechnol., 2015, 33(9): 990-995.
[94] LUNDBY A, SECHER A, LAGE K, et al. Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues[J]. Nat. Commun., 2012, 3: 876-886.
[95] ZIELINSKI R, PRZYTYCKI P F, ZHENG J, et al. The crosstalk between EGF, IGF, and Insulin cell signaling pathways-computational and experimental analysis[J]. BMC Syst. Biol., 2009, 3: 88-98.
[96] WENG Y, CHEN W, KONG Q, et al. DeKinomics pulse-chases kinase functions in living cells[J]. Nat. Chem. Biol., 2024, 1-9.
[97] CARVER R S, STEVENSON M C, SCHEVING L A, et al. Diverse expression of Erbb receptor proteins during rat liver development and regeneration[J]. Gastroenterology, 2002, 123(6): 2017-2027.
[98] REN R, MAYER B J, CICCHETTI P, et al. Identification of a ten-amino acid proline-rich SH3 binding site[J]. Science, 1993, 259(5098): 1157-1161.
[99] SONGYANG Z, SHOELSON S E, CHAUDHURI M, et al. SH2 domains recognize specific phosphopeptide sequences[J]. Cell, 1993, 72(5): 767-778.
[100] MICHALOPOULOS G K, BHUSHAN B. Liver regeneration: biological and pathological mechanisms and implications[J]. Nat. Rev. Gastroenterol. Hepatol., 2021, 18(1): 40-55.
[101] ZHENG Y, ZHANG C, CROUCHER D R, et al. Temporal regulation of EGF signalling networks by the scaffold protein Shc1[J]. Nature, 2013, 499(7457): 166-171.
[102] MARTINS P N, THERUVATH T P, NEUHAUS P. Rodent models of partial hepatectomies[J]. Liver Int., 2008, 28(1): 3-11.
[103] PARANJPE S, BOWEN W C, MARS W M, et al. Combined systemic elimination of MET and epidermal growth factor receptor signaling completely abolishes liver regeneration and leads to liver decompensation[J]. Hepatology, 2016, 64(5): 1711-1724.
[104] BAI H, FANG C W, SHI Y, et al. Mitochondria-derived H(2)O(2) triggers liver regeneration via FoxO3a signaling pathway after partial hepatectomy in mice[J]. Cell Death Dis., 2023, 14(3): 216-229.
[105] BARD-CHAPEAU E A, YUAN J, DROIN N, et al. Concerted functions of Gab1 and Shp2 in liver regeneration and hepatoprotection[J]. Mol. Cell. Biol., 2006, 26(12): 4664-4674.
[106] GU H, NEEL B G. The "Gab" in signal transduction[J]. Trends Cell Biol., 2003, 13(3): 122-130.