Nghiên cứu xác định hệ phosphoprotein và hệ protein tiết của tế bào lympho t người bằng các kỹ thuật proteomic

VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY INSTITUTE OF BIOTECHNOLOGY NGUYỄN TIẾN DŨNG CHARACTERIZATION OF THE HUMAN TLYMPHOCYTE PHOSPHOPROTEOME AND SECRETOME USING PROTEOMIC TECHNIQUES PhD THESIS IN BIOLOGY Hanoi, 2016 VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY INSTITUTE OF BIOTECHNOLOGY Nguyễn Tiến Dũng CHARACTERIZATION OF THE HUMAN TLYMPHOCYTE PHOSPHOPROTEOME AND SECRETOME USING PROTEOMIC TECHNIQUES Speciality: Biochemistry Code: 62 42 01 16 PhD THESIS IN BIOLOGY SUPERVISORS: Dr Joaquin Abián Moñux Institute of Biomedical Research of Barcelona, Spain Prof Dr Phan Văn Chi Institute of Biotechnology, Vietnam Hanoi, 2016 Declaration of Authorship I, Nguyen Tien Dung hereby declare that this thesis is from my own The results presented in the thesis are from my own works and the works shared with my colleagues under their permission Where I have consulted the work of others, this is always clearly stated Title of the PhD thesis “Characterization of the human T-lymphocyte phosphoproteome and secretome using proteomic techniques” Hanoi, 21/09/2016 Nguyễn Tiến Dũng i Acknowledgements I would like to express my special appreciation and thanks Dr Joaquin Abian Moñux and Prof Dr Phan Van Chi for your supervisions and supports in my PhD program I would like to thank you for encouraging my research and for allowing me to grow as a research scientist Your advice on my research, my career, and my daily life are priceless I would especially like to thank Dr Montserrat Carrascal not only for your training, your support in experimental works but also for your advices, sympathy and help in my daily life in Barcelona I would also like to thank my current and former colleagues (Vanessa Casas, Roberto Pinto, Edita Bueno, Marina Gay, Oriol Vidal, Joan Villanueva, Oscar Gallardo, Mari Carmen Torres) at CSIC/UAB Proteomics Laboratory, Barcelona, Spain for all your assistance in training techniques, adapting to the new working environment and the new life in Barcelona I would also like to appreciate my colleagues at Protein Biochemistry Laboratory, Hanoi, Vietnam for your help in applying the CSIC-VAST PhD program and in writing my PhD thesis I would also like to thank Mrs Bui Thi Hai Ha, Institute of Biotechnology for your help in preparing for the thesis defence A special thanks to my family Words cannot express how grateful I am to all of you for all of the sacrifices that you have made on my behalf This work was supported by Spanish National Research Council and Vietnam Academy of Science and Technology under the bilateral project “Characterization of the human primary T-lymphocyte phosphoproteome and secretome using proteomic techniques” - 2012VN0003 Hanoi, 21/09/2016 Nguyễn Tiến Dũng ii CONTENTS Declaration of Authorship i Acknowledgements ii CONTENTS iii LIST OF TABLES vii LIST OF FIGURES viii ABBREVIATIONS x INTRODUCTION Chapter BACKGROUND 1.1 Protein phosphorylation 1.1.1 Functions and roles of protein phosphorylation 1.1.2 Protein phosphorylation detection 1.1.2.1 Traditional biochemical detection methods 1.1.2.2 Mass spectrometry-based detection method 1.2 Phosphoproteomics .8 1.2.1 Phosphopeptide enrichment 1.2.2 Mass spectrometry-based quantification 11 1.2.3 Bioinformatics and data analysis 13 1.3 T cell signaling and phosphoproteomics .15 1.3.1 T cell signaling 15 1.3.1.1 Overview of T cell receptor signaling 15 1.3.1.2 Roles of phospholipase C-gamma1 in T cell receptor signaling 16 1.3.2 Phosphoproteomic studies of T cells 17 1.3.2.1 Phosphoproteomic studies of primary T cells and LymPHOS 17 1.3.2.2 Analysis of T cell receptor signaling using human Jurkat T cells 19 1.4 Secretome and phosphosecretome analysis 20 1.4.1 Secretome and protein secretory pathways 20 iii 1.4.2 Study on secretome 22 1.4.2.1 General strategy for large-scale secretome analysis 22 1.4.2.2 Phosphorylation study of secretome 22 1.4.2.3 Secretome analysis of T cells 24 1.5 Proteomic research in Vietnam 25 Chapter MATERIALS AND METHODS .27 2.1 Samples and materials 27 2.2 Methods .29 2.2.1 Methods for phosphoproteome analysis of human primary T cells and update of the LymPHOS database 29 2.2.1.1 Human primary T cell isolation and activation 29 2.2.1.2 Protein extraction and digestion 30 2.2.1.3 Peptide labeling with isobaric tags 32 2.2.1.4 Separation of peptides by Strong cation exchange chromatography 32 2.2.1.5 Phosphopeptide enrichment 33 2.2.1.6 LC-MSn analysis 33 2.2.1.7 Database search and quantitative analysis 34 2.2.1.8 Database and web application structure 35 2.2.1.9 Biological analysis 36 2.2.2 Methods for phosphoproteome analysis of human Jurkat T cells 36 2.2.2.1 Sample preparation 36 2.2.2.2 LC-MSn analysis 37 2.2.2.3 Database search and quantitative analysis 38 2.2.2.4 Biological analysis 38 2.2.2.5 Western blot analysis of LCK 39 2.2.3 Methods for phosphosecretome analysis of human Jurkat T cells 39 2.2.3.1 Sample preparation 39 2.2.3.2 LC-MSn analysis 40 iv 2.2.3.3 Database search and quantitative analysis 41 2.2.3.4 Bioinformatics analysis of phosphosecretome 41 Chapter RESULTS 42 3.1 Characterization of the human primary T cell phosphoproteome – an update of the LymPHOS database .42 3.1.1 Characterization of the human Primary T cell phosphoproteome 43 3.1.1.1 Qualitative and quantitative analysis 43 3.1.1.2 Panther-based GO classification 44 3.1.1.3 Chromosome-based classification 46 3.1.2 LymPHOS2.0 – update of the LymPHOS database 47 3.1.2.1 LymPHOS: structure and general information 47 3.1.2.2 LymPHOS: protein and peptide views 51 3.2 Phosphoproteome analysis of human Jurkat T cells 55 3.2.1 Global qualitative analysis 57 3.2.2 Global quantitative analysis 61 3.2.3 Biological analysis 63 3.2.4 Phosphorylation of LCK 66 3.3 Phosphosecretome analysis of human Jurkat T cells 70 3.3.1 Identification of proteins and phosphoproteins in the conditioned medium of Jurkat T-cells 71 3.3.2 Biological process–based classification of potential secreted phosphoproteins 72 3.3.3 Quantitative analysis of secreted phosphoproteins 73 Chapter DISCUSSION 74 4.1 Role of the LymPHOS database 74 4.2 Qualitative analysis of human Jurkat cell phosphoproteomes: contribution to the phosphosite database 76 4.3 Quantitative analysis of human Jurkat T cell phosphoproteomes: possible roles of PLCγ1 78 4.4 Human Jurkat T cell phosphoproteome: biological analysis 79 v 4.5 The role of new P-sites of LCK 82 4.6 Characterization of secreted phosphoproteins from human Jurkat T cells 84 CONCLUSIONS AND PERSPECTIVES 90 Conclusions 90 Perspectives 91 PUBLICATIONS RELATED TO THE PhD THESIS 92 REFERENCES 93 TÓM TẮT LUẬN ÁN 118 vi LIST OF TABLES Table Title Page Table 1.1 Phosphopeptide enrichment methods Table 2.1 List of chemicals and enzymes 27 Table 2.2 List of instruments and apparatuses 28 Table 2.3 List of software and tools 28 Table 3.1 General qualitative and quantitative information of human primary T cell phosphoproteome 44 Table 3.2 Quantitative data included in the LymPHOS database 50 Table 3.3 Collection of new P-sites detected from unique phosphopeptides in Jurkat T cells 59 Table 3.4 P-sites of LCK detected in the JE6.1 and Jgamma1 cell clones 67 Table 3.5 Secreted phosphoproteins verified by both SignalP and GOA 71 Table 3.6 List of secreted protein containing regulated P-site 73 vii LIST OF FIGURES Figure Title Page Figure 1.1 Overview of protein phosphorylation Figure 1.2 Phosphorylation detection in large-scale phosphoproteomics studies Figure 1.3 General phosphoproteomics workflow Figure 1.4 Overview of MS-based quantitative proteomics approaches 12 Figure 1.5 Overview of TCR signaling pathways 16 Figure 1.6 Classical and nonclassical secretion of cardiokines 21 Figure 1.7 General secretome analysis workflow 23 Figure 3.1 The experimental workflow for the charaterization of human primary T cell phosphoproteome and update of LymPHOS 43 Figure 3.2 Gene Ontology classification of the LymPHOS proteins compared to the Human Proteome 45 Figure 3.3 Chromosome localization - based classification of human primary T cell phosphoproteins 47 Figure 3.4 The website interface of the LymPHOS database (lymphos.org) 48 Figure 3.5 Schema of the LymPHOS2.0 database 49 Figure 3.6 Comparison of the Fold-change (fc) values between LymPHOS tools and DanteR 51 Figure 3.7 Protein view of LymPHOS database 52 Figure 3.8 Peptide view - quantitative summary of LymPHOS 53 Figure 3.9 The workflow for the phosphoproteome analysis of human Jurkat T cells 56 Figure 3.10 LC-MSn identification and quantification of DLLSDLQDIsDSER ER 57 Figure 3.11 Phosphopeptide and phosphoprotein sets identified in JE6.1 and Jgamma1 clones 58 Figure 3.12 Regulated phosphopeptides detected across time course in the two Jurkat cell clones 62 viii Uniprot Protein name AC Time (min) No Phosphopeptide 133 AAVLsDsEDEEK Q96ST2 Protein IWS1 homolog U 134 P05412 Transcription factor AP-1 cAMP-dependent protein kinase type II-alpha regulatory subunit Calcium/calmodulin-dependent protein kinase U type IV D Antigen KI-67 U U 139 LAsPELER RVsVCAETYNPDE EEEDTDPR ASRDPsPIQDGNED MK AQSLVISPPAPsPR IACKsPPPESVDTPT STK ASsLNFLNK 140 RSsELLVR Q9NSK0 Kinesin light chain 141 ALINsMDQNMFR Q04759 Protein kinase C theta type 142 ANsLEPEPWFFK P06239 Tyrosine-protein kinase Lck 143 YVIsDEEEEDDD Q8WVC0 RNA polymerase-associated protein LEO1 144 146 RPtLTTFFGR P16150 Leukosialin ETPHsPGVEDAPIA Q9UHB6 LIM domain and actin-binding protein K SNsLSK P53671 LIM domain kinase 147 AGGPTtPLsPTR P20700 Lamin-B1 148 ATsSSSGSLSATGR Q03252 Lamin-B2 149 153 RHsEVETDSK Q9NX58 Cell growth-regulating nucleolar protein DRSsPPPGYIPDEL Mitogen-activated protein kinase kinase Q9Y2U5 HQVAR kinase Mitogen-activated protein kinase kinase TPsEIQFHQVK Q12851 kinase kinase TTSRsPVLSR Mitogen-activated protein kinase kinase O95819 kinase kinase TTsRsPVLSR 154 ELSSPIsPK P78559 155 ASsSGSASK Q15555 156 RDsFDDR P43243 157 GPVsPSVSFQPLAR Q7Z434 158 VNsMVAYK 159 TIsAPVVRPK O15151 RNsCNVGGGGGGF K Q7L2J0 TSsKSEAGAR 135 136 137 138 145 150 151 152 160 161 162 163 164 165 166 ESGNsQELAR AQTGPLRSsLEESP FEA GAGATSGsPPAGR N GAGATsGSPPAGR N HTDDEMTGyVATR P13861 Q16566 P46013 15 Q9H0B6 Kinesin light chain 30 60 120 U U U U U U D D D U U D U U U U U U U U U U U U D D D U U D D U U U U U U U U D U D D D D D D D D Microtubule-associated protein 1A D Microtubule-associated protein RP/EB family U member Matrin-3 D D D U U U Mitochondrial antiviral-signaling protein Mitotic spindle assembly checkpoint protein MAD2A Protein Mdm4 U 7SK snRNA methylphosphate capping enzyme U D D D Q86W50 Putative methyltransferase METT10D U U U Q13257 Q86V88 Magnesium-dependent phosphatase P16455 Methylated-DNA protein-cysteine methyltransferase Q16539 U Mitogen-activated protein kinase 14 D U D D D D D U U U D D D No Phosphopeptide Uniprot Protein name AC Time (min) 15 171 AtSNVFAMFDQSQI QEFK ATsNVFAMFDQSQ IQEFK RGPTSsNPR LPLsPPLVEDSAFE PSRK TGKTEDDSsPK 172 GEVPPKEtPK P49006 173 WHQPPPsPLPLR Q68DK7 Male-specific lethal homolog D 174 LPEASQsPLVLK Q13330 Metastasis-associated protein MTA1 U 175 AAsDDGSLK KGAGDGsDEEVDG K GsFTLLWPSR LIsVEPRPEQPEPDF R LSQSSQDSsPVR sSGsETEQVVDFSD R KTSDANEtEDHLES LICK AQLsPGIYDDTSAR NDARsPGSISYLPS FFTK TCsLPDLSK TSsLPPLDWPLPAH FGQCELK SQDATFSPGSEQA EKsPGPIVSR TSPGsPR Q92614 Myosin-XVIIIa P35579 Myosin-9 B0I1T2 Myosin-Ig O95251 Histone acetyltransferase MYST2 Q09161 Nuclear cap-binding protein subunit O75376 Nuclear receptor corepressor 167 168 169 170 176 177 178 179 180 181 182 183 184 185 186 187 188 D O14950 P40692 DNA mismatch repair protein Mlh1 O96007 Molybdopterin synthase catalytic subunit O60669 Monocarboxylate transporter MARCKS-related protein U U U U D D D D D U U U U U U U U U D D D U U U U U U U U U D U D D U U Q05586 Glutamate [NMDA] receptor subunit zeta-1 D O00567 Nucleolar protein 56 P06748 Nucleophosmin P57740 Nuclear pore complex protein Nup107 Q9H1E3 Nuclear ubiquitous casein and cyclindependent kinases substrate P19338 Nucleolin QAAsPLEPK Q14980 Nuclear mitotic apparatus protein SQGDEAGGHGED Q9NZT2 Opioid growth factor receptor RPEPLsPK YLsFTPPEKDGFPS GTPALNAK Q13177 Serine/threonine-protein kinase PAK IISIFSGTEKGsK 197 U Q86WB0 Nuclear-interacting partner of ALK 194 196 D Q96PY6 Serine/threonine-protein kinase Nek1 Nuclear factor of activated T-cells, Q12968 cytoplasmic GLsEDTTEETLK 195 D U 193 191 D D 192 190 D 60 120 Myosin regulatory light chain 12B LIAHAGsLTNLAK CGsGPVHISGQHLV AVEEDAEsEDEEEE DVK QPDISCILGTGGKs PR TSTsPPPEKSGDEGs EDEAPSGED LELQGPRGsPNAR 189 30 U U U D D D D U U U U U U D D U U U U D D U U D Uniprot Protein name AC No Phosphopeptide 198 AGPVsPGCVK Q53GL7 Poly [ADP-ribose] polymerase 10 199 MIFEGPNKLsPR O94913 200 SGLTVPTsPK Q53EL6 GHTAsESDEQQWP Q9NTI5 EEK QPsEEEIIK Q15121 201 202 203 Time (min) 15 U U Pre-mRNA cleavage complex protein Pcf11 U U U Programmed cell death protein Sister chromatid cohesion protein PDS5 homolog B Astrocytic phosphoprotein PEA-15 U RAsFAEK U 211 GsVSDEEMMELR 212 GSVsDEEMMELR 213 207 208 U U U 210 206 U D 209 205 60 120 U HGEsAWNLENR P18669 Phosphoglycerate mutase ESAsPTIPNLDLLE AHTK EsASPTIPNLDLLE O75151 PHD finger protein AHTK KGsDDAPYSPTAR Q8WWQ AQsYDIQAWK PH-interacting protein PHD and RING finger domain-containing GRECsPTSSLER Q9P1Y6 protein RGFsDSGGGPPAK Q9H307 Pinin 204 30 U U U U U U U U U U U U U U U U U U U U U U U U U U U U Q15149 Plectin P13796 Plastin-2 KCsQTQCPR P29590 Protein PML 214 DLAVPAALtPR Q8TEM1 Nuclear pore membrane glycoprotein 210 215 GLTWIAHsR Q7Z3K3 Pogo transposable element with ZNF domain 216 GILtLK A5A3E0 POTE ankyrin domain family member F 217 O94906 Pre-mRNA-processing factor P25788 Proteasome subunit alpha type-3 P28066 Proteasome subunit alpha type-5 U Q8NDX1 PH and SEC7 domain-containing protein D 221 IQQQFsDLKR ESLKEEDEsDDDN M RItSPLMEPSSIEK GGPFWPQVTLNsQ DRDER DYGNsPLHR 222 EDLSPAFDHsPNK P17706 223 RLSsFVTK 224 AAsAIYR 225 P27708 Q9UKM9 Isoform of RNA-binding protein Raly -2 Q9UKM9 RNA-binding protein Raly 229 STAVTTSsAK TRDDGDEEGLLTH sEEELEHSQDTDA Q9UKM9 RNA-binding protein Raly DDGALQ AKsIVFHR P62826 GTP-binding nuclear protein Ran ALsHQEPMVSTQP APR Q86YV0 RAS protein activator like-3 RVLsAPPK 230 TAVIPINGsPR 218 219 220 226 227 228 P26599 P06400 Polypyrimidine tract-binding protein Tyrosine-protein phosphatase non-receptor type CAD protein Retinoblastoma-associated protein U U U U D U U D D U U U U D D D D U U U U U U U U U U D Uniprot Protein name AC No Phosphopeptide 231 IPGGNIYIsPLK 232 240 ISEGLPtPTKMtPR IPGGNIYIsPLKsPY K TLQTDSIDSFETQRt PR ISEGLPTPTKMtPR IPGGNIYIsPLKSPY K SLsPVAAPPLREPR Q8NDT2 Putative RNA-binding protein 15B GLVAAYSGEsDsEE P98175 RNA-binding protein 10 EQER YRsPYSGPK Q14498 RNA-binding protein 39 IGLPHsIK 241 SFsSPENFQR Q9Y580 RNA-binding protein 242 HSTPsPTR P49792 243 SEsWER O43598 244 RQSsSYDDPWK Q96D71 245 NKPLsPIK P35251 E3 SUMO-protein ligase RanBP2 Deoxyribonucleoside 5'-monophosphate Nglycosidase RalBP1-associated Eps domain-containing protein Replication factor C subunit 246 sSSPELVTHLK Q07960 Rho GTPase-activating protein 247 SEsESK Q52LW3 Rho GTPase-activating protein 29 248 RDsFDNCSLGESSK VPLAPITDPQQLQL sPLK AALLKAsPK VEAKEESEEsDED MGFGLFD VEAKEEsEESDED MGFGLFD KEESEEsDDDMGF GLFD KEEsEESDDDMGF GLFD Q5UIP0 255 YSPTsPK P24928 256 258 QSAPsPTR Q13905 AAGISLIVPGKsPT P47736 R LTEGCsFR P42677 259 LNsSENGEDR Q7L4I2 260 IPLVRsFADIGK Q6U841 261 TSGsKER 262 KSQsPSPK 233 234 235 236 237 238 239 249 250 251 252 253 254 257 Time (min) 15 30 60 120 D D D D D D D D D D D D D D D D D U U U U U D D D D U D D D U U U U U U U U D D P50914 Telomere-associated protein RIF1 D Ribonucleoside-diphosphate reductase subunit M2 60S ribosomal protein L14 P05388 60S acidic ribosomal protein P0 P31350 U U U D U U P05386 U U U U U U U U U U U U D D D D D D D D 60S acidic ribosomal protein P1 DNA-directed RNA polymerase II subunit RPB1 Rap guanine nucleotide exchange factor D Rap1 GTPase-activating protein U 40S ribosomal protein S27 D D U U Q15424 Arginine/serine-rich coiled-coil protein Sodium-driven chloride bicarbonate exchanger Scaffold attachment factor B1 U Q99590 Protein SCAF11 U U U D U U No 263 Phosphopeptide KLsPTEPK Uniprot Protein name AC O14828 267 IYHLPDAEsDEDED Q15019 FKEQTR TMIIsPERLDPFAD GGK O75533 RWDQTADQTPGAt PK SGsISVK Q8TF01 268 TPsASNDDQQE 269 EMPQDLRsPAR 270 FLMPEAYPSsPR 271 RsWDQQIK 272 QPGIHPKtPNK SAsPDDDLGSSNW O00193 EAADLGNEER 264 265 266 273 U U D D D D D D D Splicing factor, arginine/serine-rich 18 Small glutamine-rich tetratricopeptide repeatcontaining protein alpha O43166 Signal-induced proliferation-associated 1-like protein Q14493 Histone RNA hairpin-binding protein U U D U U U Smith-Magenis syndrome chromosomal region candidate gene protein-like Transcription activator BRG1 278 EVDYSDsLTEK P51532 RPDHSGGsPsPPTSE Q8TAD8 Smad nuclear-interacting protein PAR SDsQQAVK Q15036 Sorting nexin-17 RSDsQQAVK 279 RYsDFEWLR O60493 Sorting nexin-3 280 ESDQTLAALLsPK P18583 Protein SON 281 283 GSLGIsQEEQ Q9NZD8 Maspardin AQTLPTSVVTITSE Q01082 Spectrin beta chain, brain SsPGKR TVsASStGDLPK P78362 Serine/threonine-protein kinase SRPK2 284 SVsGsPEPAAK 285 SVSGsPEPAAK 286 TAsPPPPPK 287 TRHSPTPQQsNR 288 U U Small acidic protein 275 282 60 120 Splicing factor 3B subunit AISAPTsPTR 277 30 Septin-2 274 276 15 Secretory carrier-associated membrane protein O43765 Q9NQG6 Time (min) U U U D D U U U U D D D D D U U U U D U U U U U U D Q8IYB3 Serine/arginine repetitive matrix protein U U U APQTssSPPPVR U U U 289 ESPsPAPKPR D D D 290 SPsPAPPPR U U 291 SESDsSPDSK 292 AQSGsDSsPEPK U 293 AQSGSDssPEPK U 294 SGsSPEVK 295 SATRPSPsPER MSCFSRPSMSPtPL DR ALPQtPRPR 296 297 D Q9UQ35 Serine/arginine repetitive matrix protein D D D D D D D D D D D Uniprot Protein name AC Time (min) No Phosphopeptide 298 SGsSPEVDSK 299 SLSGSsPCPK D 300 D 302 SLsGSSPCPK QGSITSPQANEQSV tPQRR sGSsPGLR U U U U 303 SESDssPDSK U U U U 304 SGssPEVDSK U U U U 305 SGSEssVDQK U U U 306 VPsPTPAPK 307 309 GQSQTsPDHR DKFsPFPVQDRPES SLVFK QSHSSsSPHPK 310 SGSESsVDQK 311 SSSAsSPEMK 312 SRsWTSPK 313 RDGSYGsGR 314 RDGsYGSGR 315 GRDSPYQsR 301 308 15 30 60 120 D D D D D D U U U U U U D O75494 Serine/arginine-rich splicing factor 10 Q08170 Serine/arginine-rich splicing factor U U D D D D U U U U U U 317 U U U 318 AsGQAFELILsPR U U U 319 RAsGQAFELILsPR P16949 U U U 320 DLsLEEIQKK U 321 ESVPEFPLsPPKK U 322 KPSGGsSR 323 325 KPSGGSsR HGGVCAPAAVATs Transforming acidic coiled-coil-containing PPGAIPK Q9Y6A5 protein ILsPSMASK 326 SPVVSGDTsPR 327 SPVVsGDTsPR VPssDEEVVEEPQS R TIGGGDDsFNTFFS ETGAGK LsVDYGK AYHEQLsVAEITN ACFEPANQMVK IMNTFSVVPsPK MsMKEVDEQMLN VQNK 324 328 329 330 331 332 333 Q13242 U Serine/arginine-rich splicing factor Double-stranded RNA-binding protein GSSPtPPCsPVQPSK Q9NUL3 Staufen homolog DLsLEEIQK 316 P35269 P10636 Stathmin General transcription factor IIF subunit Microtubule-associated protein tau U U U U Tubulin alpha-1B chain U U U U U U U U U U U U U U U U U Q9C0C2 182 kDa tankyrase-1-binding protein P68363 U U U U U U U U U U U U U U P04350 Tubulin beta-4 chain U U U U P07437 Tubulin beta chain U U U U Uniprot Protein name AC No Phosphopeptide 334 NISSSPsVEsLPGGR Q92609 335 AALAPAKEsPR 336 EAASGTtPQK 337 338 TBC1 domain family member Q13428 Treacle protein HFAsPSK Q92664 Transcription factor IIIA VFIDQNLsPGK Q14186 Transcription factor Dp-1 339 TPVSGSLKsPVPR Q8NI27 THO complex subunit 340 SRsGEGEVSGLMR 341 SRSGEGEVsGLMR STAPSAAASASAS AAASsPAGGGAEA LELLEHCGVCR AADRLPNLSsPSAE GPPGPPSGPAPR VPDEEENEEsDNE KETEK TQMAEVLPsPR KAsGsENEGDYNP GR KASGsENEGDYNP GR NREEEWDPEYtPK Q13263 Transcription intermediary factor 1-beta Q13263 Transcription intermediary factor 1-beta O60784 Target of Myb protein P11388 DNA topoisomerase 2-alpha 342 343 344 345 346 347 348 Q02880 SsFSITR 351 SGSAHGsGK 352 SGsAHGSGK 353 RSPsPYYsR 354 RPEGPGAQAPSsPR P40222 355 AQsEAEK D D D D D D D D D D D U U U U U U U U D U U U U U U U U U U D D U P62995 O75643 AsGEMASAQYITA P54578 ALR AAPPPPPPPPPLEsS Q5T4S7 PR U Transformer-2 protein homolog beta Alpha-taxilin U5 small nuclear ribonucleoprotein 200 kDa helicase E3 ubiquitin-protein ligase UBR4 359 THPYsPK P22415 Ubiquitin fusion degradation protein homolog Upstream stimulatory factor 360 P08670 Vimentin 365 ISLPLPNFsSLNLR TRPGsFQSLSDALS DTPAK RPEsPSEIsPIK RPEsPSEIsPIKGSV R AGTGyASPDRTHV LAAGK AIGSTSKPQEsPK 366 AKSPTPDGsER Q96MU7 YTH domain-containing protein U U U U U U U U U U U U Ubiquitin carboxyl-terminal hydrolase 14 Q92890 364 D D FVAFSGEGQsLR 363 60 120 DNA topoisomerase 2-beta 358 362 30 D 350 361 D Thyroid hormone receptor-associated protein Q9Y2W1 AsAVSELsPR 357 15 D 349 356 Time (min) U D D D D D Q9H9C1 VPS33B-interacting protein D D U Q7Z5K2 Wings apart-like protein homolog O43379 WD repeat-containing protein 62 P18887 DNA repair protein XRCC1 U D D D D U No Phosphopeptide 367 AKSPtPDGSER 368 SEPHsPGIPEIFR Uniprot Protein name AC Time (min) 15 30 U U U 374 Q8ND82 Zinc finger protein 280C U Zinc finger CCCH domain-containing protein HSAIsPK Q8N5P1 AsLEDAPVDDLTR U Q7Z2W4 Zinc finger CCCH-type antiviral protein KFTYLGsQDR Zinc finger CCHC domain-containing protein IHsPIPDMSK Q6NZY4 U ATGDGssPELPSLE Q9H2Y7 Zinc finger protein 106 homolog R RSsPPPPPSGSSSR Q5VUA4 Zinc finger protein 318 375 NPFRGsPK 376 SPAGsPELR 377 KPGPPLsPEIR 378 IHsGEKPFK 369 370 371 372 373 Q14966 Zinc finger protein 638 Q96JM3 Zinc finger protein 828 P17027 Zinc finger protein 23 The lower case indicates the phosphorylation residue U: Up-regulation; D: Down-regulation Uniprot AC: Uniprot accession number The kinases containing regulated P-sites are marked by gray fill U 60 120 U U D D U U U U U D D D D D U U U U U U U D D D Supplemental table 3.3 Phosphopeptides and proteins containing regulated P-sites in Jgamma1 No Phosphopeptide GsFTLLWPSR RLIsVEPRPEQPEPDFR SSFFSDRGSGsR Uniprot Protein name AC Time (min) 15 30 60 120 U B0I1T2 Myosin-Ig U U O00571 ATP-dependent RNA helicase DDX3X AT-rich interactive domain-containing GPsPSPVGsPASVAQSR O14497 protein 1A VsGSFPEDSSK O15400 Syntaxin-7 26S proteasome non-ATPase regulatory MKQEGsAR O43242 subunit SRsWTSPK O75494 Serine/arginine-rich splicing factor 10 QNsLGSNEEK U AQsEAEK 10 DsVLTSK 11 RDsVLTSK 12 IMNTFSVVPsPK P04350 Tubulin beta-4 chain 13 AAQPGsGK P05455 Lupus La protein 14 ILEQsGEWWK U 15 U 17 ANsLEPEPWFFK P06239 Tyrosine-protein kinase Lck MVRPDNCPEELyQLM R SLVsKGTLVQTK P10412 Histone H1.4 18 KAtQASQEY P16104 Histone H2A.x U 19 QGsLAMEELK P16150 Leukosialin D 20 ASGQAFELILsPR 21 RAsGQAFELILsPR 22 AsGQAFELILsPR 16 O75554 WW domain-binding protein U5 small nuclear ribonucleoprotein 200 O75643 kDa helicase O75937 DnaJ homolog subfamily C member D U U U U U U U U U U D U U D P16949 Stathmin D D High mobility group protein HMGP17096 I/HMG-Y Probable ATP-dependent RNA helicase P17844 DDX5 P18887 DNA repair protein XRCC1 23 SsQPLASK 24 LLQLVEDRGsGR 25 27 AIGSTSKPQEsPK GLsEDTTEETLKESFD P19338 Nucleolin GSVR LAQYEsK P24534 Elongation factor 1-beta 28 SRsAQEPAR P25685 DnaJ homolog subfamily B member 29 TDsSPNQAR 30 GLPPsMER 31 GRDVESVQtPSK P26599 Polypyrimidine tract-binding protein Heterogeneous nuclear ribonucleoprotein P38159 G P46013 Antigen KI-67 32 RVQPQWsPPAGTQPCR P49589 Cysteinyl-tRNA synthetase, cytoplasmic 26 D D U U D U D D D U U D U D U U No Phosphopeptide Uniprot Protein name AC 36 SKHEEEEWtDDDLVES P51946 Cyclin-H L MGsGVER Heterogeneous nuclear ribonucleoprotein P52272 MGLsMER M GCGVVKFEsPEVAER 37 AKsIVFHR 38 SGSAHGsGK 39 RSPsPYYSR 40 AVFVDLEPtVIDEVR P68363 Tubulin alpha-1B chain 41 YRPGtVALR P68431 Histone H3.1 42 GSsDVDQLGK Q00534 Cyclin-dependent kinase 43 RDGSYGsGR Q08170 Serine/arginine-rich splicing factor 44 LTWHsCPEDEAQ 45 SLsDsESDDSK 46 AYSSFGGGRGsR 47 RASSSGsASK 48 AAGGAPsPPPPVR 33 34 35 P62826 GTP-binding nuclear protein Ran Time (min) 15 30 60 120 U D D D U U U P62995 Transformer-2 protein homolog beta U U U U U U U U U Q13185 Chromobox protein homolog D Q15056 Eukaryotic translation initiation factor 4H U Microtubule-associated protein RP/EB Q15555 family member Q4KMP TBC1 domain family member 10B D D D 54 IINLGPVHPGPLsPEPQ Q86T03 Transmembrane protein 55B PMGVR AAQQQQPSAsPR Q86U86 Protein polybromo-1 Isoform of Ribonucleoprotein PTBLLsPLSSAR Q8IY67 binding Serine/arginine repetitive matrix protein SVSGsPEPAAK Q8IYB3 Serine/arginine repetitive matrix protein TRHsPtPQQSNR TQLsQGR Q8ND56 Protein LSM14 homolog A 55 TAsGSSVTSLDGTR Q92597 Protein NDRG1 D 56 SsVVFADEK U 57 ILSQStDSLNMR 58 AAQAGRQsSAK Q92608 Dedicator of cytokinesis protein Rho guanine nucleotide exchange factor Q92974 Q99986 Serine/threonine-protein kinase VRK1 59 GAEETSWsGEER Q9H7N4 Splicing factor, arginine/serine-rich 19 RPLFLAPDFDRWLDEs Q9P035 3-hydroxyacyl-CoA dehydratase DAEMELR PHD and RING finger domainKENPsPLFSIK Q9P1Y6 containing protein STAVTTSsAK TRDDGDEEGLLtHSEE ELEHSQDTDADDGAL Q9UKM RNA-binding protein Raly Q TRDDGDEEGLLTHsEE ELEHSQDTDADDGAL Q 49 50 51 52 53 60 61 62 63 64 U U D U U U D D U U D U U U U U U No Phosphopeptide Uniprot Protein name AC Apoptotic chromatin condensation inducer in the nucleus Isoform B of Ras GTPase-activating protein-binding protein 67 Q9UKV STtPPPAEPVSLPQEPP Q9UN86 KPR -2 SGsSPEVK 68 SGsSPEVDSK 69 SGsESsVDQK Serine/arginine repetitive matrix protein 70 SSSAsSPEMK 65 66 71 FTRsQEEAR GTYVPSsPTR Q9UQ35 Time (min) 15 30 60 120 U D U WD repeat domain phosphoinositideQ9Y4P8 interacting protein The lower case indicates the phosphorylation residue U: Up-regulation D: Down-regulation Uniprot AC: Uniprot accession number The kinases containing regulated P-sites are marked by gray fill U U U U D D Supplemental table 3.4 Potential secreted phosphoproteins detected in conditioned media of human Jurkat T cells Uniprot AC A5A3E0 B0I1T2 S-1010 O14745 S-280 O14950 O15511 S-77 Actin-related protein 2/3 complex subunit O43491 Band 4.1-like protein O60343 TBC1 domain family member O95084 S-58 S-588 S-570 S-138 No P-sites Protein names S-733 POTE ankyrin domain family member F Unconventional myosin-Ig Na(+)/H(+) exchange regulatory cofactor NHERF1 S20 (1) x Myosin regulatory light chain 12B Serine protease 23 P00558 S-203 Phosphoglycerate kinase 10 P02787 S-306 Serotransferrin 11 P04075 T-49, S-39 12 P04406 13 x Fructose-bisphosphate aldolase A x S-333 Glyceraldehyde-3-phosphate dehydrogenase x P05771-2 T-641 Lupus La protein 14 P06239 15 P06493 16 P06733 Y-505 T-14, Y15, Y-160, T161, T-166 S-37, S-40, Y-44, S-419 17 P07910 S-233 18 P08238 S-226, S-255 19 P08575 20 Tyrosine-protein kinase Lck x Cyclin-dependent kinase Alpha-enolase Heterogeneous nuclear ribonucleoproteins C1/C2 Heat shock protein HSP 90-beta x S-973 Receptor-type tyrosine-protein phosphatase C x P09651 S-6 Heterogeneous nuclear ribonucleoprotein A1 21 P10412 S-104 Histone H1.4 22 P10809 S-70 60 kDa heat shock protein, mitochondrial 23 P13612 S-1021, S-1023 24 P13639 T-54 25 P13796 S-257, S-323, S-5, S-7 26 P13861 S-99 27 P14618 28 x x x Integrin alpha-4 Elongation factor x x S-37 Plastin-2 cAMP-dependent protein kinase type II-alpha regulatory subunit Pyruvate kinase PKM P16104 S-140 Histone H2AX 29 P16150 30 P16949 31 P17612 S-355, T-341 S-16, S-25, S-46, S-31, S-38 T-196, T-198, T-202, Y205 x Leukosialin Stathmin x cAMP-dependent protein kinase catalytic subunit alpha x 32 Uniprot AC P17987 33 P18669 S-14, Y-119 34 P19338 T-121, S-67, S-563, T-76 35 P21291 S-192 36 P21333 S-2152, S-2158, S-2163 37 P22102 Y-348, T-349 38 P22234 S-27 Filamin-A Trifunctional purine biosynthetic protein adenosine-3 Multifunctional protein ADE2 39 P22314 S-835 Ubiquitin-like modifier-activating enzyme x 40 P23396 T-220, T-221, S-224 40S ribosomal protein S3 x 41 P23528 S-3, S-41 Cofilin-1 x 42 P25325-2 S-15 43 P25787 Y-6, S-7, S-9, T-11 Proteasome subunit alpha type-2 x 44 P25788 S-250 Proteasome subunit alpha type-3 x 45 P27448 T-211, S-215 46 P27708 47 P27816 48 P27824 S-1859 S-787, S-330, T-332, S507, T-521, S-358, S941, S-1073 S-583 49 P30086 T-51, S-52, S-54 50 P33316 S-99 51 P34932 52 No P-sites Protein names (1) S-551 T-complex protein subunit alpha x Phosphoglycerate mutase x Nucleolin x Cysteine and glycine-rich protein x x x x DNA replication licensing factor MCM3 MAP/microtubule affinity-regulating kinase CAD protein Microtubule-associated protein x Calnexin x x S-647 Phosphatidylethanolamine-binding protein Deoxyuridine 5'-triphosphate nucleotidohydrolase, mitochondrial Heat shock 70 kDa protein P35251 S-368 Replication factor C subunit 53 P35579 S-1943 Myosin-9 x 54 P35580 S-1956 x 55 P36507 T-394 56 P36871 T-115, S-117 Myosin-10 Dual specificity mitogen-activated protein kinase kinase Phosphoglucomutase-1 57 P37837 S-237 Transaldolase x 58 P38159 S-352 RNA-binding motif protein, X chromosome x 59 P40222 S-514 Alpha-taxilin 60 P48634 S-1219 Protein PRRC2A 61 P49006 T-148, S-22, T-85 MARCKS-related protein 62 P49207 S-12 60S ribosomal protein L34 63 P50395 S-61 Rab GDP dissociation inhibitor beta 64 P50914 S-139 60S ribosomal protein L14 65 P51858 S-165 Hepatoma-derived growth factor x 66 P53396 S-481, S-455 ATP-citrate synthase x 67 P54578 S-143, S-148 Ubiquitin carboxyl-terminal hydrolase 14 x x x x x 68 Uniprot AC P60174 69 70 No P-sites Protein names (1) S-58 Triosephosphate isomerase x P60709 S-60 Actin, cytoplasmic x P61978 S-284, S-216 Heterogeneous nuclear ribonucleoprotein K x 71 P62826 S-135 GTP-binding nuclear protein Ran x 72 P63241 S-46 Eukaryotic translation initiation factor 5A-1 x 73 P68363 Tubulin alpha-1B chain x 74 Q01082 Spectrin beta chain, non-erythrocytic x 75 Q01130 T-41, S-48 S-2340, S-2341, S-2138, S-2102, T-2107 S-191, S-26, T-22, T-25 Serine/arginine-rich splicing factor x 76 Q01196 S-212 77 Q01813 S-386 78 Q01968 S-156 79 Q06210 S-261, T-262, T-263 80 Q07955 S-199, S234, S-238 81 Q07960 S-50, S-51 82 Q14004 T-1246 Cyclin-dependent kinase 13 83 Q14103 T-127 Heterogeneous nuclear ribonucleoprotein D0 84 Q14204 S-4368 85 Q14247 S-418, S-417, Y-421 86 Q14847 T-104 LIM and SH3 domain protein 87 Q15019 S-218 Septin-2 x 88 Q15185 S-113 Prostaglandin E synthase x 89 Q15365 Poly(rC)-binding protein x 90 Q16181 Septin-7 x 91 Q16555 S-173, S-189, S-190 T-332, S-334, S-424, T426 T-509, T-514 Dihydropyrimidinase-related protein x 92 Q53ET0 S-433 93 Q6IAA8 Y-5, S-7, S-12 94 Q6KC79 S-2658 95 Q6UX27 S-169 96 Q6UXC1 S-138, S-139 97 Q6ZSZ5 S-1103 Rho guanine nucleotide exchange factor 18 98 Q7Z460 S-600 CLIP-associating protein 99 Q7Z6Z7 S-1907 E3 ubiquitin-protein ligase HUWE1 100 Q86YV0 S-72, S-51 101 Q86YZ3 Y-1349 102 Q8IX21 S-586, S-590, S-591 103 Q8IZP0 S-183, S-225 Runt-related transcription factor ATP-dependent 6-phosphofructokinase, platelet type Inositol polyphosphate 5-phosphatase OCRL-1 Glutamine fructose-6-phosphate aminotransferase [isomerizing] Serine/arginine-rich splicing factor x x x Rho GTPase-activating protein x x Src substrate cortactin CREB-regulated transcription coactivator Ragulator complex protein LAMTOR1 Nipped-B-like protein V-set and transmembrane domain-containing protein Apical endosomal glycoprotein RAS protein activator like-3 Hornerin Protein FAM178A Abl interactor 104 Uniprot AC Q8ND76 105 Q8TCD5 S-182 5'(3')-deoxyribonucleotidase, cytosolic type 106 Q92598 S-809 Heat shock protein 105 kDa 107 S-1706, S-1784, S-1791 109 Q92608 Q96BY63 Q96FV2 110 Q96G03 S-165 Phosphoglucomutase-2 111 Q96JY6 S-197 PDZ and LIM domain protein 112 Q9BUH6 S-148, T-145, S-148 113 Q9BUQ8 S-14 Probable ATP-dependent RNA helicase DDX23 114 Q9BY44 S-506 Eukaryotic translation initiation factor 2A 115 Q9H3Z4 S-10 DnaJ homolog subfamily C member 116 Q9H501 S-153 ESF1 homolog 117 Q9NR45 S-275 Sialic acid synthase x 118 Q9NVS9 S-241 Pyridoxine-5'-phosphate oxidase x 119 Q9UNE7 S-19 E3 ubiquitin-protein ligase CHIP 120 Q9UQ80 S-363 Proliferation-associated protein 2G4 121 Q9UQN3 122 Q9Y2V2 S-199 S-30, S-32, S-41, T-45 123 Q9Y2W1 S-243, S-248, S-698, S682, S-684, T-685, T874, S-253, Y-54, S-55 124 Q9Y512 S-294 125 Q9Y5K6 S-224 No 108 P-sites Protein names S-326 Cyclin-Y S-13 S-55, T-50, T-52 Uniprot AC: Uniprot accession number x Dedicator of cytokinesis protein SH3 domain-containing kinase-binding protein Secernin-2 x Protein PAXX x x Charged multivesicular body protein 2b Calcium-regulated heat stable protein x Thyroid hormone receptor-associated protein x Sorting and assembly machinery component 50 homolog CD2-associated protein (1), compared with Bonzon-Kulichenko et al., 2011 x indicates proteins in common (1) [...]... IMAC T (Thr) Threonine TCR T cell receptor TEMED Tetramethylethylenediamine TFA Trifluoroacetic acid TMT Tandem mass tag TOF Time of flight V Volt Y (Tyr) Tyrosine xi 1 INTRODUCTION Phosphorylation is one of the most common and important reversible posttranslational modifications (PTMs) regulating protein functions Phosphorylation and dephosphorylation result in conformational changes in the structure... costimulation of the CD3/TCR complex and other costimulatory receptors such as CD28 or CD4, lymphocytespecific protein tyrosine kinase (LCK) is attracted to the intracellular tails of the TCR-CD3 complex where it phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) of the intracellular CD3 domains These phosphorylation events facilitate the phosphorylation of ζ-chain associated protein. .. developed software (adapted from Kanshin et al., 2012) 10 Table 1.1 Phosphopeptide enrichment methods (Engholm-Keller and Larsen, 2013) Method Principle Main properties IMAC Electrostatic interaction with multivalent metal ion Sensitive to salts and detergents MOAC Electrostatic interaction with metal oxide High specificity, highly robust toward salt and detergents pTyr- Selective enrichment of pTyr imunoprecipitatio... BACKGROUND 1.1 Protein phosphorylation 1.1.1 Functions and roles of protein phosphorylation Phosphorylation is among the most important and well-studied posttranslational modifications (PTMs) of proteins; about one-third of all human proteins was estimated as phosphoproteins (Cohen, 2001) Protein phosphorylation is mostly occurred at serine (Ser), threonine (Thr) and tyrosine (Tyr) residues even though there... detection methods such as Western blot (Mandell, 2003) However, successful detection is highly dependent on the specificity and affinity of the antibody corresponding to the phosphoprotein of interest 1.1.2.2 Mass spectrometry-based detection method Mass spectrometry is a powerful analytical technique, enabling not only the detection of phosphopeptides/phosphoproteins but also the specific location of their... of the TCR, formation of the F-actin cup, inside-out activation of integrins, polarization of microtubules, production of cytokines, and alternative splicing of messenger RNA 20 Furthermore, the authors suggested that modulation of protein- protein interactions (PPIs) related to the changes in serine and threonine phosphorylation state was appropriate to different signaling systems (Mayya et al., 2009)... allow the discovery of new, physiologically relevant kinases as well as the understanding of the mechanisms involved in the regulation of protein function This information is the first step towards the design of new therapies based on drugs controlling either kinase/phosphatase activities or the activity of other proteins in key points of the affected pathways T lymphocytes (T cells) play a vital role... the radioactivity is released with a list of the protein predicted peptides to identify those that contain serine, threonine, or tyrosine at the same position in the peptide sequence (Sickmann and Meyer, 2001; MacDonald et al., 2002) Two-dimensional gel electrophoresis (2-DE) has been also used to detect phosphoproteins (Peck et al., 2006) The replacement of the neutral hydroxyl groups on serine, threonine... about 3000 proteins The quantitative data, presenting hundreds of regulated P-sites, gave important new insights on the protein phosphorylation profile of effector T cells (Navarro et al., 2014) The LymPHOS database LymPHOS is a web-oriented database containing peptidic and protein sequences and spectrometric information on the phosphoproteome of human primary T cells (Ovelleiro et al., 2009) LymPHOS... TCR-activation through the plasma membarne complex 4 Our work presented the first collection of secreted phosphoproteins from human Jurkat T (the Jurkat E6.1 clone) cells with 125 potential secreted phosphoproteins were identified and verified Among them, five proteins contained regulated P-sites in response to Jurkat T cell activation with PMA/ionomycin: PA2G4, ALDOA, THRAP3, DOCK2, and stathmin 4 Chapter 1