蛋白质修饰位点预测详解

1、蛋白质组学蛋白质修饰位点分析目录实验目的2实验平台2实验过程3一、"人类connexin43”蛋白质磷酸化位点修饰 31、"人类connexin43”蛋白质序列下载 32、uniprot数据库查看蛋白磷酸化位点 43、在线软件预测指定蛋白磷酸化位点 6（1） DISPHOS 1.3预测未知蛋白磷酸化位点 6（2） PhosphoSitePlus预测指定蛋白磷酸化位点 114、"人类connexin43”蛋白质磷酸修饰结论 14二、“人类血红蛋白”糖基化位点修饰 151、N型糖基化位点预测 152、O型糖基化位点预测 18（1 ）哺乳动物 O型糖基化位点预测 18（

2、2）真核生物O型糖基化位点预测 203、uniprot数据库查看蛋白质糖基化修饰位点 224、“人类血红蛋白”糖基化位点修饰结论 22实验结论23（特别提示:ctrl+单击目录下的标题链接，可以跟踪标题；ctrl+单击标题后的图标可以返回目录）实验目的找出“人类connexin43 ”蛋白质上面的所有可能磷酸化位点,并说明为什么（注释）找出“人类血红蛋白”上面的糖基化位点，注释结果实验平台uniprot 数据库：/ （查看蛋白的修饰情况）预测未知蛋白磷酸化位点DISPHOS: /disphos/Phos

3、phoSitePlus预测未知蛋白的糖基化修饰位点N型糖基化位点预测：http:/www.cbs.dtu.dk/services/NetNGlyc/O型糖基化位点预测：http:/www.cbs.dtu.dk/services/NetOGlyc/ http:/www.cbs.dtu.dk/services/Yi nOYan g/实验过程一、“人类conn ex in 43 ”蛋白质磷酸化位点修饰1、“人类connexin43”蛋白质序列下载蛋白序列：fasta .txtProtein)connexin43 AND "Homo sapi

4、ens1!搜索关犍词Save search Advanced进入NCBI的 Protein数据库r)g£： 0 Summary . 20 per page Sorted by Default order19Results: 1 to 20 of 45of 3REcNmm已 Full二Gao lunctionDotEin; AltName Full二ConneM3; Short二CM3: A附Full二Gbd junction 43 kD nEsrt DotEin Homo 宮apiens382 aa proteinAccession: P17302 2 G: 117706陽 nPED

5、t FAST AGnDliics ReI 呂 tEd S 电口 UEncES选择序列gao iumtion mlphe-l potein Homo 与apimns382 aa proteinAccession: NP_Q00156,1 Gl： 4504001GenPecrt FASTA G阳Mies Related SequencesIdentic白I Frot它ins uDiQiiilim isoform 2 Homo sanimns3581 aa proteinAccession： NP_001291271 1 Gl： 747811S02%护訥 FAST离 G呂訂hies RglNtEd

6、S电口liemes Identical Protgiris2、uniprot数据库查看蛋白磷酸化位点网站链接：http:/www .uni /connexin43援盂蛋白质名称UniProtKBBLAST Align Retneve/TD mappingResultsFilter by2勿 Reviewed (40) Swiss-ProtUnreviewed (148)TrEMBLPopular organismsHuman (13)Mouse (10)Rat (9)Bovine (3)Zebrafish (3)Other organismsEntryP18246口 P2324

7、2土 DownloadEntry naCXA1 BOVINCXA1_MOUSEF ColumnsProtein namesGap junction alpha-1 protein口 P08050CXA1_RAT|昜Gap junction alpha'lGjprotein口 P17302CXA1_HUMAN霜Gap junction alpha-1 proteinGJGap junction alpha-1 protein057474CXA1_-DANRE賠Gap junction alpha-1 proteingjsoP14154CXA1_-CHICK希Gap junction al

8、pha-1 proteinGJP16B63CXA1_-XENLA豐Gap junction alpha-191Amino acid modifications磷酸位点查看Disulfide bond154 j 192Cross-llink1Disulfide bond1144 - 144187 198Cross-link1237 -237Modified residue12斗7 - 2斗7Modified residue1255 -255Modified residue1262 -262Modified residue1271 -271Modified residue:314 -314Modi

9、fied residue1325 -325Modified residue1323 -328Modified residue1330 -330Modified residue1344 -344Modified residue1365 -36SModified residue1368 - 368Modified residue1369 -369Modified residue*373 -373Feature keyPosition(s) Length 1 PublicationGlycyl lysine isopeptide (Lys-Gly) (interchain with G-Cter i

10、n SUMO) 1 Publication Glycyl lysine isopeptide (Lys-Gly) (interchain with G-Cter in SUMO； llPhosphotyrosme By sjmj|anty氨酸Phosphoserine 2 Publications -尹丝氨酿Phosphoserine * 2 PublicaboAS/3、在线软件预测指定蛋白磷酸化位点(1) DISPHOS 1.3预测未知蛋白磷酸化位点网站链接：/disphos/开始预测InformationAboyt Usap 巳 REfmr

11、ences Datasets® Eukaryotes诜择物种人类OrganismFunctional CategoryDISPHOS 1.3Disorder-Enhanced PhosphorylationSites PredictorPaste your FASTA formatted protein sequeiice可以将序列粘帖入业文本框Or select a file:G：僵白质组学非业蛋白叮23Q4o刘4卜王红*畑射txt|汶悦O Default PredictorGroup PredictorsGroup predictors are trained to minimi

12、ze the number of total misclassified resic qroup (see Usmcie for detailed instructions)Kingdoms and Viruses'*'H. sapiens O M. muse ulus O R non/egicus O C. elegans _ S cerevisiae_ D melanogasteiC>A. thahanaCj regulation (?can 匚即 C J cytoskeleton (/'membrane O ri bosomalO inhibitorsCj

13、transport(') kinasesC： degradation ：biosynthesisO metabolism GPCRsO ArchaeaO Bacteria,-OV,ruf厂开始预测Predict 吏盖结果DISPHO由蛋白序列预测蛋白磷酸修饰位点，总共有 37个丝氨酸 修饰位点，13个苏氨酸修饰位点，16个酪氨酸修饰位点。但是根 据打分，只有8个丝氨酸修饰位点可能存在。Statistics丝氨酸璘酸化修饰位点数目Number of phosphorylated serines:NumJoer of phosphorylatedNumber of phosphorylat

14、ed tyroncutof37(21outof13(0outof16(0.622%)000%)000%)PositionResidueScore5S0 06017Y001418S0 02819T001127S002639T0 00843S0 04050S003856T0 02566Y0.03069S003173s0 01786s0 00989T001092Y0 00998Y0 021118T0 008137Y0 018154T0 003155Y0 002158S0 027163S0 033175Y0 003177Y0.002180S0 014182S0 008185Y0 005186T0 02

15、2201S0 035204T0 007207T0 006217S0 025SequenceYes/NoMGDVfSALGKE77QAYSTAGvqaystagg247Y0 354QAYSTAGGK250T0 053KTA7LSVLFZ251S0 234匚匚LGTAVES255s0 998TAVESAWGD262s1 000GDZQSAFRC265Y0 140FRCKTQQPG267Y0 116EKVCYDKSF272S0 124CYDKSFPIS273S0 233SFPZSHVrtF275T0 221rrrvsvPTL279S0 402VSVFTLLYL282S0 498PTLLYLAHV28

16、6¥0 347AHVF'/VMRK290T0 217KVAQTDGVN296S0 264KKFKYGIEE297S0 091G 匚 I.RTYIIS301Y0 053rLRTYZISI306S0 146TYZZSZLFK313Y0 049:LFKSIFEV314s0 086325s0 143LIQV：YTYGF326T0 019QWYIYGFSL328S0.095IYGFSLSAV330s0 104GFSLSAVYT344s0.095LSAVYTCKR364s0 889SAVYTCKRD365s0 935DCFLSRPTE368s0 964LSRPTEKTZ369s0 974

17、PTE5：TZFZ：372s0 977MLT7SLVSL373s0 984KSDPPYHAM可BSGAJL KDCG GSQKQKYAFNGC MGU莘 CSSPTAPLLSPM SPPG YKLVHNNSSCRKN罠gV7ANYGQAG QAGSGSTZTZSNDNQN DQRP QRPSSSRASRASSSRASRAS(2) PhosphoSitePlus预测指定蛋白磷酸化位点网站链接：Home开始预测FhosphotjiteKlus"with grant support frtwnN1AAA 1 IHomePhosphoSite

18、FAwtth gAdvanced S arch/ Browse Fu'xtions:PhosphoSitePlus* (PSP) is an onHne 吁哼tern% biologj" tesourte picdmg comprehen$ and tools for the grudy of pro teiir post - traiislat lonal modificatiDfis (PTMs) tncludtng d ubiquitinatton, acetylation and tnethylation. See 出bout PhosphoSitE above to

19、r tndPlea&e cite the following reference fbi thi5 resource: Hornbeck PV et al (2015) Phosph muialirts, PTMi and recalibraiions. Nucleic ?dds Res 4j:D512-2D.Search F asutts for:conn；xin43j® Modification-specific Antibc2Protein*specific AntibodiesPROTEIN OR SUBSTRATE SEARCHProtein Name: %conn

20、exin43l1PROTEINGJAl connexin43GJjProtein, Sequencej or Reference SearchSite SearchDec 2011 Download 'ThospJanuary 2012 issue of NucleicJul 2011 Multiple SequenceJui 2011 Download PvMOLComparative Site SearchCITE «TATIQTIC结果筛选参考文献来源多于5篇的磷酸化修饰位点，丙氨酸磷酸化修饰位点数目为1、丝氨酸为17、苏氨酸数目为1、酪氨酸数目为7Modificati

21、on Sites and Domains点击只查看磷酿修饰位点Click hEGJA1 (human) 一 382 amino acids |j Hide sites with only 1 MS/'HTP reference |_)Show only sites with rr050100150200250A257 :S25E S；K9S5K109K10SY137K136K134K133K128 K1441 IIIL1S251 Y2ET250Y261 ¥247S244 S262K243K258SIl I II ill 1 U I*匚 onnexinTMTM Connexin

22、 匚匚匸 TMTMModification Srtes and DomainsClick heGJAl (human) 382 amino acids |_ Hide sites with only 1 MS/HTP reference Show only sites with r根据文献来源筛选磷酸位点Connexin扎丙氨酸 &丝氨酸 T：苏氨醱 ¥：络氨酸TF1 匚onnexin CCC TMA257S25E £S251 ST250 Y2iY247 Y26S24E S262I I! II II IB100150200250蛋白质组学Click heModifi

23、cation Sites and DomainsGJAL (human) - 382 amino acids _ Hide sites with only 1 MS./HTP reference £ Show only sites with rY137Connexm TMA:1 S:17T:1Y:7TM Connexin CCC TM¥2iA257S25E ¥247 ¥26S24J S262I H III20150200250蛋白质组学33Show Multiple Sequence AlignmentS5 /£LJ1 "kGRLLD

24、 霹酸修饰位£egeS244KDRVKGKsDPyHAtsY247VKGKsDPyHAtsGALT250KsDPyHAtsGALsPAS251sD Py HA.t sGALsPAKS255HA七 sGAL8PAKDCGsA257tsGALsPARDCGsQRS262sPARDCGsQRjAyFN¥265KDCGsOK/AyFNGCs127CGsQKyAyFNGCssPS272yAiFNGCssPtAPLsS273AyFNGCssPtAPLsPT275FMGCssPtAPLsPMsS279ssPtAPLsPMsPPGjS282tAPLsPMaPPGyRLV¥286s

25、PMsPPGyKLVtGDRT290PPGKLVtGDRHllssS296tGDRNNssCRNyNKS297tGDRJINssCRNNKa¥301NNssCRNtHKOAsEQS306RIIyNKQAsEQNWANyY313SEQNWANYSAEQNRMSQ列fZJIysAEQNRMGUJS14UQAGS tl SrlSnAT326BUGQAGstlsNsEAaS328GQAGstlsHsHAQPF5330GstlsNsHAOPFDFU341PFDFPDONQNsEKLAS344FPDDNQNsKKLAAGHS364AIVDORPssRAssRAS365IVDQRPs sRAssR

26、AsS368QRPssRAssRAssRFS369RPssRAssRAssRPR5372sRAs gRAssRPRPDDS373KAssRAs sRPRPDD L4、“人类connexin43”蛋白质磷酸修饰结论在线数据库查询和在线软件预测结果基本一致，“人类 conn ex in43 ”蛋白质磷酸位点数量相对一般蛋白质较多，且丝氨酸磷 酸修饰位点最多。“人类connexin43 ”蛋白质极有可能是一种活性较高的蛋白质，与机体的生物活性显著相关。通过各种文献阅读和网络筛选发现 connexin43蛋白质是一种哺 乳动物连接蛋白，是主要的细胞缝隙连接蛋白，其表达的异常与多种 疾病的发生有关。二、

27、“人类血红蛋白”糖基化位点修饰Ncbi下载人类血红蛋白的蛋白序列保存为：hemoglob in .txt1、N型糖基化位点预测网站链接：http:/www.cbs.dtu.dk/services/NetNGlyc/开始预测NetNGIyc 1.0 Server人工神经网络方祛预测蛋白“端糖基化修饰The NetNglyc server predict NGytosyi戲ion sitms in hiimmn proteins |jsing artificial neural networks that examinekrstructionsOiriDut formatSUBMISSIONPas

28、te a single sequence or several sequences <n 匚/SP format into the field below:贴入蛋白质序列/IGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHTOPENFRSubmit a file in MSN format directly from your local d賦 选挥g件|未选择文件Afternattveiy. type in Swiss-Prot fD/AC (e g CBG_HUMAW)朗 Generate graphics Show actditional thresholds

29、(032,0.75t 0.90) in the graph(s)By default, predictions are done only on the Asn*Xaa-Ser/Thr sequons (ind Asn-Pro-SerTtir) Predict on all Asn residues use this only if you know whm you are doing!I SubmitClear fields结果Asn- X aa- 3 e r / Thr sequons in the sequence out put below are highlighted in blu

30、e. Asp ar ag ines predicted to be K-glycosylated are highligh in red.Output for 'SequenceTaming: This seq口enct say not cont ain a signal peptide ! !Proteins without signal peptides are unlikely to be exposed 'to the IT-fflycosylation Bachinery and thus ay not be glycosylated (in vivo) even t

31、hough they cont ain pot ent ial sot ifs一般耒说没有信号駄是无祛预测N型翟SignalP-O euk predictions are as仇1""所臥最后预测结果星有疑问的t na*eCsax pos ?Yksk pos ?Ssaxpos ?Ssean ?D?Sequence0.28527 R0.0S227 N0.3103 N0.088 N0.075NSicnalP output is explained atcbg. dtu dk/器霍n理.P/output#尊*需尊#雷尊P需尊*累尊#需#*#*#P*#*#P*尊#*車希*專#*享希

32、#总尊#丰尊#总尊#尊尊#牯#您#E#專#尊E*#專#声專*需#*樹NaMe： SequenceLength： 1471VHLTPEEKSAVTALIGKVJIVDEVGGEALGRLLWYPITQRFFESFGDLSTPBAV1GHPEVKAHGEKVLGAFSDGLAHLD MLKGTFATL SELHCD KLHVDF EHFRLLGNVL ?C¥LAHHFGXEF TPP TQAAYQSTVAG VANALAHKYHn.n (Threshold=0 5)高于05的打分刀 一共钊E位詈极有可 卜睡点修饰能由N型糖基4Potentia1 Threshold 化修饰-1 111 M

33、etHG 1 yc 1.6： predluted N-fl 1 yccsy 1 atiort sites in Sequence0.50.250ioa回140SeqUTasePositionPot ent i aliJury igreeBeSequence20 NVDE0. 8209(9/S)Seqiuence58 HPKVCL 6789(9/9)S6(iiience81 HLEG0- 7600(9/9)Sequence103 SFRL0-4814(5/9)Sequence109 SVLV0. 7492(9/3)Sequence140 SALA0.4504(8/S)H-Glyc resultV

34、ARNIITG: PRO-X1.红色点是可能 的糖基化位点Spfliija rsr ntn « i + i ah2、O型糖基化位点预测(1 )哺乳动物0型糖基化位点预测网站链接：http:/www.cbs.dtu.dk/services/NetOGIyc/开始预测NetOGtyc 4.0 Server血测哺乳动物的o型糖羞The NetOglyc server produces neural network predictions ofrnucin type GalNAcO-glycosylation sites in mammalianView the 的 sior, histc,

35、 of this serverSUBMISSIONPaste a single sequence or several sequences tn : format into the field below： MVHLTPEEK3AVTALWKWDEVG（；EAL（；RLLWYPWTQRFFESF（；DLSTPDAWCNPKVKAHGK K VLGAF SDGL AHLD 肛 KG TF ATL SELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEF TPPVQAAYQ EWAGVOALAHKYH/结果CENTERFO R B!0 LQ GI CALSEQU ENCEANA LYS

36、IS CBSNetOGIyc 4.0 Server prediction resultsTechnical University of Denmark#source-version NetOGlyc 4* U* CL 13 #date 15-5-26tsegnanesource feature st arterudSEQUENCEnetOGlyc-4. 0. 0. 13CARBOHYDSEQUENCEnetOGlyc-4,0. 0,13CARBOHYDSEQUENCEnetOGlyc-1. CL （L 13CARBOHYDSEQUENCEnetOGlyc-l. CL 0* 13CAKEOHYD

37、SEQUENCEnet0&lyc-4» CL CL 13CAREOHYDSEQUENCEnetOGlyc-4» 0. 0.13CARBOHYDSEQUENCEnetOG lyc-4. 0*0 13CAREOHYDSEQUENCEnet0Glyc-4. 0* 0. 13CARBOHYLSEQUENCEnetOGlyc-4. 0. 0-13CAREOHYDSEQUEM3EnetOGlyc-4,0.0,13CAREOHYDSEQUENCEnetOGlyc-4.0. 0.13CAEEOHYDSEQUENCEnetOGlyc-d» 0. 0.13CAEBOHYD#T

38、ype Proteinstrand.0 3 950135511345578frame5101339455051T3858890124D. 143阳O型糖基化1 D*455663 |.0.3T9134.0.09T470B0.29484 ”.0.213S8T.0.323743.0.134485.0.0771958.0,179965.0. 13062 .0.181412.(2 )真核生物0型糖基化位点预测网站链接：http:/www.cbs.dtu.dk/services/Yi nOYang/开始预测二丿YinOYang 1,2预测真核生物的o型糖基化检点The YinOYang 讥fWW serv

39、er produces neural network predictions for O-C-GIcNAc attachment sites in eukar/oticprote markpossi bl phosphorylated sites and hence identfy 'Yin-Yang'1 sites.SUBMISSIONFmste a jingle sequence or several sequences in 二你审 format into the field below AFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLV

40、CVLAHHFGKEFTPPVQAAYQKV-j VAGVANvALAHKYH/Submit a file m format directiy from your local disk:选择文件丨未选择立件Submft Uniprot ID/AC:S generate graphics yin-yang site predictions (i.e. cross-NetPhos scans)Output®' show only positive sitesC show all S/T residuesNetPho: thresd£ a胡卩 is autamaticaliy run on al! sequences A warning is displayed if a signal peptide is predictedSubmitClear fields结果SequenceLength： 147VHLTPEEKSA¥rALTGKVHVDEVGGEALGRLLWYPTTQRFFESFG