蛋白質(zhì)修飾位點(diǎn)預(yù)測(cè)詳解

1、 蛋白質(zhì)修飾位點(diǎn)分析 卻錄 實(shí)驗(yàn)?zāi)康?. 2 實(shí)驗(yàn)平臺(tái) . 2 實(shí)驗(yàn)過(guò)程 . 3 一、人類 connexin43蛋白質(zhì)磷酸化位點(diǎn)修飾 . 3 1、人類 connexin43蛋白質(zhì)序歹 U 下載 . 3 2、 uniprot 數(shù)據(jù)庫(kù)查看蛋白磷酸化位點(diǎn) . 4 3、 在線軟件預(yù)測(cè)指定蛋白磷酸化位點(diǎn) . 6 (1) . DISPHOS 1.3 預(yù)測(cè)未知蛋白磷酸化位點(diǎn) . 6 (2) . PhosphoSitePlus 預(yù)測(cè)指定蛋白磷酸化位點(diǎn) . 11 4、人類 connexin43蛋白質(zhì)磷酸修飾結(jié)論 . 14 二、“人類血紅蛋白糖基化位點(diǎn)修飾 . 15 1、 N 型糖基化位點(diǎn)預(yù)測(cè) . 15 2、 O

2、 型糖基化位點(diǎn)預(yù)測(cè) . 18 (1) . 哺乳動(dòng)物。型糖基化位點(diǎn)預(yù)測(cè) . 18 (2) . 真核生物。型糖基化位點(diǎn)預(yù)測(cè) . 20 3、 uniprot 數(shù)據(jù)庫(kù)查看蛋白質(zhì)糖基化修飾位點(diǎn) . 22 4、 “人類血紅蛋白糖基化位點(diǎn)修飾結(jié)論 . 22 實(shí)驗(yàn)結(jié)論 . 23 (特別提示：ctrl+單擊目錄下的標(biāo)題鏈接，可以跟蹤標(biāo)題； ctrl+單 擊標(biāo)題后的圖標(biāo) W可以返回目錄) 實(shí)驗(yàn)?zāi)康? 找出“人類connexin43 蛋白質(zhì)上面的所有可能磷酸化位點(diǎn), 并說(shuō)明為什么(注釋) 找出“人類血紅蛋白上面的糖基化位點(diǎn)，注釋結(jié)果 實(shí)驗(yàn)平臺(tái)拶 uniprot 數(shù)據(jù)庫(kù)： :/ / (杳看蛋白的

3、修飾 情況) 預(yù)測(cè)未知蛋白磷酸化位點(diǎn) DISPHOS: :/ /disphos/ PhosphoSitePlus :/ 預(yù)測(cè)未知蛋白的糖基化修飾位點(diǎn) N 型 糖 基 化 位 點(diǎn) 預(yù) 測(cè)： :/ cbs.dtu.dk/services/NetNGlyc/ O 型 糖 基 化 位 點(diǎn) 預(yù) 測(cè)： :/ cbs.dtu.dk/services/NetOGlyc/ :/ cbs.dtu.dk/services/YinOY ang/ 實(shí)驗(yàn)過(guò)程 一、“人類connexin43 蛋日質(zhì)磷酸化位點(diǎn)修飾 * 1、“人類connexin43蛋白質(zhì)序列下載

4、* 蛋白序列： fasta.txt t(2) Protein 進(jìn)入NCBI的 connexin43 AND Homo sapiens 搜素關(guān)鍵詞 Save search Advanced Protein數(shù)據(jù)庫(kù) _ Ings: 0 Summary, 20 pet page, Sorted by De M3; Short二CK43: A See GE (8NNENIN43)_q奕川心門 口Qtein 加h目 1 in the Gene database connexin43 reference sequences TrmnscriDt PEteiri Results: 1 to 20 of 45

5、RecNam巳 Full二Gan junction alpha-1。己in： AHName Full二Connq 1 Full二Gap iunction 43 kDa heart DQtein Hemo spiwns / 382 aa protein / Accession: P17302 2 Gl: 117706 , G-nP 的 FAST A GaJhics Rwlmt 臼 d Svcju色 news ItjentiBjgToteins qar iunction alph*1 DrotBin HOITIH sapiens 2 382 aa protein Accession: NP_000

6、156 1 Gl: 4504001 GwPept FASTA GrDhics Related SguenmA Identical Pi麗ns ubiQuilirM iEorm 2 Homo $a iens 3 581 aa protein Accession： NP_001291271 1 Gl： 747811802 G-nP如t FASTA Graphics RelatM S如LIEFIES Identical Proteins 2、uniprot數(shù)據(jù)庫(kù)查看蛋白磷酸化位點(diǎn) * 網(wǎng)站鏈接: :/ / :/ / connexin43 搜索蛋白質(zhì)名稱 U

7、niProtKB BLAST Align Retneve/TD mapping Results Filter by Reviewed (40) Swiss-Prot Unreviewed (148) TrEMBL Popular organisms Human (13) Mouse (10) Rat (9) Bovine (3) Zebrafish (3) Dther organisms 二二M i Download Add to basket 丸 Columns % BLAST Entry Entry na Protein names P18246 P23242 P08050 CXA1 BO

8、VIN CXA1 MOUSE CXA1 RAT 口 P17302 CXA1_ .HUMAN 057474 CXA1_ _DANRE P14154 CXA1_ .CHICK P16B63 CXA1_ _XENLA Gap junction alpha-1 protein Gap junction alpha-1 protein Gap junction alpha-1 proMn Gap junction alpha-1 protein Gap junction alpha-1 protein Gap junction alpha-1 protein Gap junction alpha-1 g

9、j so gj 蛋白質(zhì)組學(xué) 網(wǎng)站鏈接: :/ /disphos/ 6 Amino acid modifications 磷酸位點(diǎn)查看 Disulfide bond1 54 i 192 Cross-link1 144 一 144 Disulfide bond1 187 i 198 Cross-link1 237 - 237 Modified residue1 247 - 247 Modified residue1 255 - 255 Modified residue1 262 - 262 Modified residue1 271 - 271 Modified re

10、sidue: 314 - 314 Modified residue1 325 - 325 Modified residue1 32& - 328 Modified residue1 330 - 330 Modified residue1 344 - 344 Modified residue1 365 - 355 Modified residue1 368 - 368 Modified residue1 369 - 369 Modified residue1 373 - 373 Position(s) Length 1 1 1 1 1 1 1 1 Publication Glycyl l

11、ysine isopeptide (Lys-Gly) (interchain with G-Cter in SUMO) 1 Publication Glycyl lysine isopeptide (Lys-Gly) (interchain with G-Cter in SUMO) 1 Phosphotyrosine - Qy similarity、客氨酸 Phosphoserine 2 Publications 絲氨酸 1 Phosphoserine . 2 Publicatio 1 SFitnJsgy砒&門8四smlmriG 半胱氨酸 Phosphoserine * j Publi

12、cation Phosphoserine; by 8# 1 Publication - Phosphoserine; by CK1A. thahana 蛋白質(zhì)組學(xué) 9 Cj regulation (? cancer C J cytoskeleton (/membrane O ri bosomal O inhibitors Cj transport (J kinases C1 degradation L biosynthesis O metabolism 。GPCRs O Archaea O Bacteria,- OVinr尸開始預(yù)測(cè) Predict 史置蛋白質(zhì)組學(xué) 10 結(jié)果 DISPHO明蛋

13、白序列預(yù)測(cè)蛋白磷酸修飾位點(diǎn)，總共有 37個(gè)絲氨酸 修飾位點(diǎn)，13個(gè)蘇氨酸修飾位點(diǎn)，16個(gè)酪氨酸修飾位點(diǎn)。但是根 據(jù)打分，只有8個(gè)絲氨酸修飾位點(diǎn)可能存在。蛋白質(zhì)組學(xué) 11 Statistics 絲氨酸磷酸化修飾位息數(shù)目 Number of phosphorylated serines: Number of phosphorylated threonines Number of phosphorylated tyron 8 cut of 37 (21-622%) 0 out of 13 (0.000%) 0 out of 16 (0.000%) Residue Score Sequence Yes

14、/No 0 060 MGDWSALGK Y 0014 S 0 028 T 0011 S 0026 T 0 008 S 0 040 S 0 038 T 0 025 Y 0.030 S 0031 S 0 017 S 0 009 T 0010 Y 0 009 Y 0 021 T 0 008 Y 0 018 T 0 003 Y 0 002 S 0 027 S 0 033 Y 0 003 Y 0 002 S 0 014 s 0 008 Y 0 005 T 0 022 S 0 035 T 0 007 T 0 006 S 0 025 S KVQA/STAG VQAYSTAGG QAYSTAGGK KLSVL

15、FI LLLGTAVES TAVESAWGD GDEQSAFRC FRCKTQQPG EKVCYDKSF CYDKSFPZS SFPISHVHF TTFVSVPTL VSVPTLLYL PTLLYLAHV AHVFWMRK KVAQTDGVN KKFKYGIHE GLLRTYIIS rLRTYIISI TYZZSILFK :LFKSIFEV LrQCYTYGF QWYIYGFSL IYGF3LSAV GFSLSAVYT LSAVYTCKR SAVYTCKRE DCFLSRPZE LSRPTEKTZ PTEKTZFI： MLWSLZSL 247 Y 0.354 KSDP 250 T 0 053

16、PYHA 251 S 0 234 K可, 255 s 0 998 SGAL 262 s 1 000 KDCG 265 Y 0 140 GSQK 267 Y 0 116 QKYA 272 S 0 124 FKGC 273 S 0 233 NGCS 275 T 0 221 CSSP 279 S 0 402 TAPL 282 S 0 498 LSFM 286 Y 0 347 290 T 0 217 YKLV 296 S 0 264 RNN 297 S 0 091 FINNS 301 Y 0 053 SCRK 306 S 0 146 NKQA 313 Y 0 049 KHAN 314 S 0 086

17、V7ANY 325 s 0 143 GQAG 326 T 0 019 QAGS 328 S 0.095 GSTZ 330 s 0 104 TZSN 344 s 0.095 DNQN 364 s 0 889 DQRP 365 s 0 935 QRPS 368 s 0 964 SSRA 369 S 0 974 SFIAS 372 s 0 977 SSRA 373 s 0 984 SRAS Position 5 17 18 19 27 39 43 50 56 66 69 73 86 89 92 98 118 137 154 155 158 163 175 177 180 182 185 186 20

18、1 204 207 217 蛋白質(zhì)組學(xué) 12 (2) PhosphoSitePlus 預(yù)測(cè)指定蛋白磷酸化位點(diǎn) 杼 網(wǎng)站鏈接: :/ 開始預(yù)測(cè) PhosphoSiteKlus Home with gnnt support fmm Advanced S arch/ Browse Fuxtions: Dec 2021 Download Thosp January 2021 issue of Nucleic Jul 2021 Multiple Sequence Jui 2021 Download PvMOL Comparative Site Search CITE TA

19、TIQTIC 結(jié)果 篩選參考文獻(xiàn)來(lái)源多于5篇的磷酸化修飾位點(diǎn)，丙氨酸磷酸化修 飾位點(diǎn)數(shù)目為1、絲氨酸為17、蘇氨酸數(shù)目為1、酪氨酸數(shù)目為7Home PhosphoSiteF A wtth g PhosphoSitePlus* (PSP) is an onHne 野鳴 terns biologj tesourte picdmg comprehen$ and tools for the grudy of pro teiir post - traiislat lonal modificatiDfis (PTMs) tncludtng d ubiquitinatton, acetylation and

20、 tnethylation. See 篇 bciut PhosphoSit舊 above tor tnd Plea&e cite the follwing reference fbi thi5 resource: Hornbeck PV, et al (2021) Phosph Nucleic ? dds Res Search F asutts for: conn；xin43 jfl Modification-specific Antibc 2Protein*specific Antibodies Gl PROTEIN OR SUBSTRATE SEARCH Protein Name:

21、，| connexin43( 姓:亍胴 SEARCH GJA1 connexin43 GJJ Protein, Sequence, or Reference Search Site Search PROTEIN 蛋白質(zhì)組學(xué) 13 Modification Sites and Domains 點(diǎn)擊只查看磷酸修飾位點(diǎn)Click hE GJA1 (human) 一 382 amino acids Hide sites with only 1 MS/HTP reference |_j Show only sites with rr A257 - S25E S； K9 K109 K10S Y137 K1

22、36 K134 K133 K128 K144 1 IIIL 1 S251 2e T250Y261 247 K織 S244 S263 K243K258S ii i iii JH i S5 * Connexin TM TM Connexin CCC TM TM Modification Srtes and Domains Click he GJAl (human) 382 amino acids |_ Hide sites with only 1 MS/HTP reference Show only sites with r 根據(jù)文獻(xiàn)來(lái)源篩選磷酸位點(diǎn) A：丙 氨釀 S：絲 氨酸 T：蘇 氨酸 Y：

23、絡(luò) 氨酸 A257 S25E S251 S T250 Y2i Y247 Y26 S24 S262 I I! M II H 0 50 100 150 200 250 Connexin TF1 Connexin CCC TFI 蛋白質(zhì)組學(xué) 14 0 50 100 150 200 250蛋白質(zhì)組學(xué) 15 Modification Sites and Domains Click he GJ Al (human) - 382 amino acids _ Hide sites with only 1 MS； HTP Y137 =凹 Show only sites with r A:1 S:17 2i T:

24、1 A257 S2SE Y:7 Y247 26 S244 S262 i i iIi Connexin TM TM TM Connexin CCC TM 100 150 2i 250 蛋白質(zhì)組學(xué) 16 Show Multiple Sequence Alignment LTP 竺竺年) hUman 0 2 2 11 0 3 D 18 5 / 躊* |JGKLLD 酸修飾位 3 9 0 7 1 7 8 254 D 5 0 4 1& 13 2 1 19 9 12 11 0 10 D 1 0 2 。1 24 2 23 3 1 12 乾44 Y24? T250 S251 S255 A257 S26

25、2 265 127 S272 S273 T275 S279 S282 286 KDRVKGKsDPyHAts VKGKsDPyHAtsGAL KsDPyHAtsGALsPA sD Py HA.t sGALsPAK HAtsGAL8PAKDCGs tsGALsPARDCGsQR sPARDCGsQRjAyFN KDCGsOK/AyFNGCs CGsQKyAyFNGCssP yAiFNGCssPtAPLs AyFNGCssPtAPLsP FMGCssPtAPLsPMs ssPtAPLsPMsPPGj tAPLsPMaPPGyRLV sPMsPPGyKLVtGDR 2 17 0 312 1 8 7

26、9 0 9 7 9 7 10 0 2 0 1 5 4 母 11 24 6 12 6 2 3 9 2 T290 PPGKLVtGDRHHss S296 tGDRNNssCRNyNK S297 tGDRJINssCRNNKa 301 NNSSCRNTHKOASEQ S306 RIIyNKQAsEQNWANy Y313 SEQNWANYSAEQNRM SQ T326 列4 s AE QNRMG 巨 g. j UK14UQAGS tl SrlSnA RMGQAGstlsNsEAQ S328 GQAGstlsHsHAQPF 5330 GstlsNsHAOPFDF U341 PFDFPDONQNsEKLA

27、 S344 FPDDNQNsKKLAAGH S364 AIVDORPssRAssRA S365 IVDQRPs sRAssRAs S368 QRPssRAssRAssRF S369 RPssRAssRAssRPR 5372 sRAs aRAssRPRPDD S373 KAssRAs sRPRPDD L 4、“人類connexin43蛋白質(zhì)磷酸修飾結(jié)論 由 在 線 數(shù) 據(jù) 庫(kù) 查 詢 和 在 線 軟 件 預(yù) 測(cè) 結(jié) 果 根 本 一 致 ， “ 人 類 connexin43 蛋白質(zhì)磷酸位點(diǎn)數(shù)量相對(duì)一般蛋白質(zhì)較多，且絲氨酸磷 酸修飾位點(diǎn)最多?！叭祟恈onnexin43蛋白質(zhì)極有可能是一種活性較 蛋白

28、質(zhì)組學(xué) 17 高的蛋白質(zhì)，與機(jī)體的生物活性顯著相關(guān)。 通過(guò)各種文獻(xiàn)閱讀和網(wǎng)絡(luò)篩選發(fā)現(xiàn) connexin43蛋白質(zhì)是一種哺 乳動(dòng)物連接蛋白，是主要的細(xì)胞縫隙連接蛋白，其表達(dá)的異常與多種 疾病的發(fā)生有關(guān)。 二、“人類血紅蛋日糖基化位點(diǎn)修飾 * Ncbi下載人類血紅蛋白的蛋白序列保存為： hemoglobin.txt 1、N型糖基化位點(diǎn)預(yù)測(cè)歸 網(wǎng)站鏈接： :/ cbs.dtu.dk/services/NetNGlyc/ 開始預(yù)測(cè)蛋白質(zhì)組學(xué) 18 NetNGIyc 1-0 Server人工神經(jīng)網(wǎng)絡(luò)方法預(yù)測(cè)蛋白囚端糖基化修飾 The NetNglyc server preUictN-Giyo)s?iat

29、iDn 引teg in human proteins |jsing artificial neural networks that examine Output format SUBMISSION Paste a single sequence or several sequences in format into the field below: GKKVLG A AFSDGLAHLDNLKGTFATLSELHCDKLHTOPENFR 貼入蛋白原序列 回 I : /I Submit a file in FAS日 format directly from your local d/sR: :選

30、擇E伴I未選擇文件 Aitemativety. type inSwiss-ProtiD/AC (e g CBG_HUMAW) M Generate graphics Show actditional thresholds (032,0.75* 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 a

31、re doing! 結(jié)果利交 _ I Submit | Clear fields 蛋白質(zhì)組學(xué) 19 Asn- X aa- 3 e r / Thr sequons in the sequence out put below are highlighted in blue. Asp ar ag ines predicted to be IT-glycosylated are highlighted in red. Taming: This sequence say not cont ain a signal peptide! ! Proteins vithout signal peptides a

32、re unlikely to be exposed to the K-fflycosylation Bachinery and thus *ay not be glycosylated (in vivo) even C ax pos ? Y ax pos ? S ax pos ? Ssean ? D ? 0.285 27 R 0. 062 27 N 0.310 3 N 0.088 N 0. D75 N is explained at trttp = /TVT,(;bg- 1ation sites i n mammalian CENTERFO R BID L。GI CALSEQU ENCEANA

33、 LYSIS CBS versian 2 #source-versioi #date 15-5-26 i NetCXlyc 4 D. IL 13 #Type Protein tsegnane source feature st art end SEQUENCE netOGlyc-4.0.0-13 CARBOHYD SEQUENCE netOGlyc-4. 0. 0. 13 CARBOHYD SEQUENCE netOGlyc-3 CARBOHYD SEQUENCE netOGlyc-l. 0. Q. 13 CAKEOHYD SEQUENCE netOGlyc-4 CL CL 13

34、 CAREOHYD SEQUENCE netOGlyc-4. 0. 0.13 CARBOHYD SEQUENCE netOGlyc-4* Q. D.13 CAREOHYD SEQUENCE netOGlyc-3 CARBOHYL SEQUENCE netOGlyc-4.0.0,13 CAREOHYD SEQUENCE netOGlyc-4, 0, 0, 13 CAREOHYD SEQUENCE mtOGlyc-4. 0 0. 13 CAEEOHYD SEQUENCE netOGlyc-d CL k 13 CAEBOHYD strand. 0 3 9501358020 3 9501

35、35802 5113455788 5113455788 9 9 1 1 frame 5 10 13 39 45 50 51 T3 85 88 90 124 頃淑取電笙 g L 143830毋 糖 基 化1 0,455&63 | . 0.3T9134 . 0.09T470B 0.29484 . 0.213S8T 0.323743 . 0.134485 . 0.0771958 . 0,179965 . 0. 13062 , 0.181412 . score 蛋白質(zhì)組學(xué) 22 (2)真核生物。型糖基化位點(diǎn)預(yù)測(cè) 由 網(wǎng)站鏈接： :/ cbs.dtu.dk/services/YinOYang/

36、開始預(yù)測(cè) Yin0Yang1,2 預(yù)測(cè)真核生物的。型糖基化位點(diǎn) The YinOYang WW.； server produces neural network predictions forO-R-GIcNAcattachment sites in eukar/oticprote markpossi ble phosphorylated sites and hence identify Yin-Yang1 sites. SUBMISSION Paste a jingle sequence or several sequences in format into the field below

37、AFSDGLAHLD1ILKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKV_J VAGVAN v ALAHKYH _ / Submit a file m . format directiy from your local disk: 選擇文件|未選擇文件 Submit Umprot !D/AC: 必 generate graphics yin-yang site predictions (i.e. cross-NetPhos scans) Output show only positive sites O show all S/T res

38、idues NetPlios thresliold 0.5 瀝舊爐1 is automatically run on all sequences A warning 沽 displayed if a signal peptide is predicted Submit Clear fields 結(jié)果蛋白質(zhì)組學(xué) 23 Sequence Length： 147 VHLTPEEKSAVTALTGKVHVDEVGGEALGRLLWYPTTQRFFESFGDLSTPDAVWGNPKVXAHGKKVLGAFSDGLAHL NLKGTFATLSELHCDKLHVDPENFKLLGIIVLVCVLAHHFGEEFTPPVQAAYQEWAGVAHALAHKYB Residue O-GlcNAc Potential Thresh. Thresh- result (1) ?2 蛋白質(zhì)組學(xué) 24 3、uniprot數(shù)據(jù)庫(kù)查看