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[求助]
通過HPLC-MS-MS分析多肽序列
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大家好， 最近使用HPLC-MS-MS檢測了蛋白質(zhì)的氨基酸序列。但是苦于沒有相關(guān)學(xué)科背景，我是學(xué)食品科學(xué)專業(yè)的。人家給了結(jié)果，我完全不了解應(yīng)該怎么分析？ 有沒有相關(guān)專業(yè)的同學(xué)或老師，給一點(diǎn)指導(dǎo)和幫助，不勝感激。 我就是從一種植物糖蛋白中通過電泳實(shí)驗(yàn)，跑出了條帶。然后將條帶切割下來，通過in-gel-digestion的操作，然后，將其進(jìn)行HPLC-MS-MS分析。得出的結(jié)果，大概如下，完全不了解應(yīng)該怎么分析。 C:\Users\Lij\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.Outlook\Z433GRZQ\Peptide Summary Report (sene M1).htm C:\Users\Lij\AppData\Local\Microsoft\Windows\Temporary Internet Files\Content.Outlook\Z433GRZQ\Peptide Summary Report (seyal down1).htm Error tolerant 1. gi|136429 Mass: 24394 Score: 80 Matches: 2(0) Sequences: 2(0) emPAI: 0.14 RecName: Full=Trypsin; Flags: Precursor Check to include this hit in error tolerant search Query Observed Mr(expt) Mr(calc) ppm Miss Score Expect Rank Unique Peptide 35 421.7593 841.5040 841.5022 2.16 0 37 1 1 U R.VATVSLPR.S 417 737.7068 2210.0986 2210.0967 0.86 0 42 0.45 1 U R.LGEHNIDVLEGNEQFINAAK.I Proteins matching the same set of peptides: gi|494360 Mass: 23458 Score: 80 Matches: 2(0) Sequences: 2(0) Chain A, The Refined 1.6 Angstroms Resolution Crystal Structure Of The Complex Formed Between Porcine Beta-trypsin And Mcti-a, A Trypsin Inhibitor Of Squash Family gi|999627 Mass: 8814 Score: 80 Matches: 2(0) Sequences: 2(0) Chain B, Refined 1.8 Angstroms Resolution Crystal Structure Of Porcine Epsilon-Trypsin gi|1942351 Mass: 13284 Score: 80 Matches: 2(0) Sequences: 2(0) Chain A, Crystal Structure Of The First Active Autolysate Form Of The Porcine Alpha Trypsin gi|2914482 Mass: 23460 Score: 80 Matches: 2(0) Sequences: 2(0) Chain A, Complex Of The Second Kunitz Domain Of Tissue Factor Pathway Inhibitor With Porcine Trypsin gi|3318722 Mass: 23457 Score: 80 Matches: 2(0) Sequences: 2(0) Chain E, Leech-Derived Tryptase InhibitorTRYPSIN COMPLEX gi|110590762 Mass: 23458 Score: 80 Matches: 2(0) Sequences: 2(0) Chain A, Trypsin In Complex With Borate gi|157878102 Mass: 24157 Score: 80 Matches: 2(0) Sequences: 2(0) Chain E, Complex Of Eeti-Ii With Porcine Trypsin gi|242253868 Mass: 25865 Score: 80 Matches: 2(0) Sequences: 2(0) trypsinogen precursor [Sus scrofa] gi|315583496 Mass: 23430 Score: 80 Matches: 2(0) Sequences: 2(0) Chain A, The Bowman-Birk Type Inhibitor From Mung Bean In Ternary Complex With Porcine Trypsin -------------------------------------------------------------------------------- 2. gi|7331218 Mass: 65978 Score: 54 Matches: 2(0) Sequences: 2(0) emPAI: 0.05 keratin 1 [Homo sapiens] Check to include this hit in error tolerant search Query Observed Mr(expt) Mr(calc) ppm Miss Score Expect Rank Unique Peptide 110 517.2633 1032.5120 1032.5087 3.17 0 24 43 1 U R.TLLEGEESR.M 247 738.3960 1474.7774 1474.7780 -0.37 0 31 7 1 U R.FLEQQNQVLQTK.W Proteins matching the same set of peptides: gi|11935049 Mass: 66027 Score: 54 Matches: 2(0) Sequences: 2(0) keratin 1 [Homo sapiens] gi|50979272 Mass: 63751 Score: 54 Matches: 2(0) Sequences: 2(0) keratin, type II cytoskeletal 1 [Canis lupus familiaris] gi|119395750 Mass: 65999 Score: 54 Matches: 2(0) Sequences: 2(0) keratin, type II cytoskeletal 1 [Homo sapiens] gi|146741296 Mass: 65160 Score: 54 Matches: 2(0) Sequences: 2(0) keratin 1 [Sus scrofa] gi|160961491 Mass: 65450 Score: 54 Matches: 2(0) Sequences: 2(0) keratin, type II cytoskeletal 1 [Pan troglodytes] gi|189054178 Mass: 65980 Score: 54 Matches: 2(0) Sequences: 2(0) unnamed protein product [Homo sapiens] gi|281341811 Mass: 39324 Score: 54 Matches: 2(0) Sequences: 2(0) hypothetical protein PANDA_012419 [Ailuropoda melanoleuca] gi|296211766 Mass: 66379 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: LOW QUALITY PROTEIN: keratin, type II cytoskeletal 1 [Callithrix jacchus] gi|297262447 Mass: 65170 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1-like isoform 6 [Macaca mulatta] gi|297262449 Mass: 64014 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1-like isoform 5 [Macaca mulatta] gi|297474460 Mass: 63113 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Bos taurus] gi|338726100 Mass: 64748 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1-like [Equus caballus] gi|350583977 Mass: 65210 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Sus scrofa] gi|351695070 Mass: 65036 Score: 54 Matches: 2(0) Sequences: 2(0) Keratin, type II cytoskeletal 1, partial [Heterocephalus glaber] gi|358421409 Mass: 63127 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Bos taurus] gi|375314771 Mass: 66013 Score: 54 Matches: 2(0) Sequences: 2(0) keratin 1 [Homo sapiens] gi|375314773 Mass: 66029 Score: 54 Matches: 2(0) Sequences: 2(0) keratin 1 [Homo sapiens] gi|375314775 Mass: 65930 Score: 54 Matches: 2(0) Sequences: 2(0) keratin 1 [Homo sapiens] gi|375314777 Mass: 66086 Score: 54 Matches: 2(0) Sequences: 2(0) keratin 1 [Homo sapiens] gi|375314779 Mass: 66026 Score: 54 Matches: 2(0) Sequences: 2(0) keratin 1 [Homo sapiens] gi|375314781 Mass: 66071 Score: 54 Matches: 2(0) Sequences: 2(0) keratin 1 [Homo sapiens] gi|397522081 Mass: 65967 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: LOW QUALITY PROTEIN: keratin, type II cytoskeletal 1 [Pan paniscus] gi|402886100 Mass: 65722 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Papio anubis] gi|403296725 Mass: 66749 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Saimiri boliviensis boliviensis] gi|410964495 Mass: 64673 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Felis catus] gi|426224422 Mass: 62704 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Ovis aries] gi|426372622 Mass: 65855 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Gorilla gorilla gorilla] gi|440896124 Mass: 66445 Score: 54 Matches: 2(0) Sequences: 2(0) Keratin, type II cytoskeletal 1, partial [Bos grunniens mutus] gi|441632443 Mass: 56633 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Nomascus leucogenys] gi|472373681 Mass: 63343 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Odobenus rosmarus divergens] gi|504141816 Mass: 61167 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1b [Ochotona princeps] gi|504141818 Mass: 66065 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: LOW QUALITY PROTEIN: keratin, type II cytoskeletal 1 [Ochotona princeps] gi|507671822 Mass: 64881 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: LOW QUALITY PROTEIN: keratin, type II cytoskeletal 1 [Octodon degus] gi|511910681 Mass: 64327 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Mustela putorius furo] gi|512810918 Mass: 65800 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 isoform X1 [Heterocephalus glaber] gi|512810921 Mass: 64413 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 isoform X2 [Heterocephalus glaber] gi|513023021 Mass: 64223 Score: 54 Matches: 2(0) Sequences: 2(0) PREDICTED: keratin, type II cytoskeletal 1 [Heterocephalus glaber] gi|444731089 Mass: 126035 Score: 54 Matches: 2(0) Sequences: 2(0) Keratin, type II cytoskeletal 1 [Tupaia chinensis] 通過以上鏈接，可以調(diào)出我的結(jié)果文件。其中還有一些是以圖片的形式給出的一些score，我不曉得什么意思。 信件原文如下， Please find the results for your MS samples attached. Some identifications are clear Seyal down 2 beta-galactosidase (ASp), Seya up alpha-l-arabinofuranosidase (Asp), sene down glycine Max while others are complicated by keratin contamination. 是不是說我的樣品中含有很多角蛋白雜質(zhì)，我查了下角蛋白，只要是皮膚，毛發(fā)等造成的蛋白污染。 |
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DBWJ
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3樓2013-09-04 08:46:46
wizardfan
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【答案】應(yīng)助回帖
★
感謝參與，應(yīng)助指數(shù) +1
137167741: 金幣+1, 小木蟲交流，共同進(jìn)步~~ 2013-09-17 08:43:17
感謝參與，應(yīng)助指數(shù) +1
137167741: 金幣+1, 小木蟲交流，共同進(jìn)步~~ 2013-09-17 08:43:17
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不知道你到底要的是什么幫助？ LC-MS/MS得到多肽序列常用的有兩種，一個(gè)是搜數(shù)據(jù)庫：把所有蛋白質(zhì)都理論上酶解走M(jìn)S/MS，然后把實(shí)際得到的圖譜和這些理論的圖譜去比對，看哪個(gè)更接近，另外一個(gè)是de novo根據(jù)圖譜來推測氨基酸組成。 但是不明白你的結(jié)果里有個(gè)emPAI, 這個(gè)是定量分析，而且這個(gè)技術(shù)誤差很大。 |
2樓2013-09-04 06:11:59
sky10016
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DBWJ
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yfly520: 金幣+2, ★有幫助 2013-09-06 13:47:28
yfly520: 金幣+3, 非常感謝您的回答，很有用，我也在擔(dān)心是不是做錯(cuò)了，因?yàn)槲也恢酪话愕慕馕霭被嵝蛄�，是HPLC-MS-MS，或者TOF-MS-MS？我從結(jié)果上看到信息，是用TOF做的，看來您真的是很專業(yè)，謝謝。 2013-09-17 01:34:14
137167741: 金幣+1, 小木蟲交流，共同進(jìn)步~~ 2013-09-17 08:43:43
yfly520: 金幣+2, ★有幫助 2013-09-06 13:47:28
yfly520: 金幣+3, 非常感謝您的回答，很有用，我也在擔(dān)心是不是做錯(cuò)了，因?yàn)槲也恢酪话愕慕馕霭被嵝蛄�，是HPLC-MS-MS，或者TOF-MS-MS？我從結(jié)果上看到信息，是用TOF做的，看來您真的是很專業(yè)，謝謝。 2013-09-17 01:34:14
137167741: 金幣+1, 小木蟲交流，共同進(jìn)步~~ 2013-09-17 08:43:43
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你這個(gè)我感覺應(yīng)該是mascot檢索的結(jié)果，標(biāo)號2就是角蛋白啊。 還有就是hplc-MS/MS就得到這么點(diǎn)結(jié)果嗎？我感覺應(yīng)該得到好多結(jié)果的啊，你這個(gè)結(jié)果怎么感覺和做Maldi-tof-tof的結(jié)果似的啊 |
5樓2013-09-06 09:10:32
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