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[求助]
通过HPLC-MS-MS分析多肽序列
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大家好， 最近使用HPLC-MS-MS检测了蛋白质的氨基酸序列。但是苦于没有相关学科背景，我是学食品科学专业的。人家给了结果，我完全不了解应该怎么分析？ 有没有相关专业的同学或老师，给一点指导和帮助，不胜感激。 我就是从一种植物糖蛋白中通过电泳实验，跑出了条带。然后将条带切割下来，通过in-gel-digestion的操作，然后，将其进行HPLC-MS-MS分析。得出的结果，大概如下，完全不了解应该怎么分析。 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] 通过以上链接，可以调出我的结果文件。其中还有一些是以图片的形式给出的一些score，我不晓得什么意思。 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. 是不是说我的样品中含有很多角蛋白杂质，我查了下角蛋白，只要是皮肤，毛发等造成的蛋白污染。 |
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wizardfan
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sky10016
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4楼2013-09-04 17:27:59
DBWJ
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【答案】应助回帖
★ ★ ★ ★ ★ ★
yfly520: 金币+2, ★有帮助 2013-09-06 13:47:28
yfly520: 金币+3, 非常感谢您的回答，很有用，我也在担心是不是做错了，因为我不知道一般的解析氨基酸序列，是HPLC-MS-MS，或者TOF-MS-MS？我从结果上看到信息，是用TOF做的，看来您真的是很专业，谢谢。 2013-09-17 01:34:14
137167741: 金币+1, 小木虫交流，共同进步~~ 2013-09-17 08:43:43
yfly520: 金币+2, ★有帮助 2013-09-06 13:47:28
yfly520: 金币+3, 非常感谢您的回答，很有用，我也在担心是不是做错了，因为我不知道一般的解析氨基酸序列，是HPLC-MS-MS，或者TOF-MS-MS？我从结果上看到信息，是用TOF做的，看来您真的是很专业，谢谢。 2013-09-17 01:34:14
137167741: 金币+1, 小木虫交流，共同进步~~ 2013-09-17 08:43:43
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你这个我感觉应该是mascot检索的结果，标号2就是角蛋白啊。 还有就是hplc-MS/MS就得到这么点结果吗？我感觉应该得到好多结果的啊，你这个结果怎么感觉和做Maldi-tof-tof的结果似的啊 |
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DBWJ
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yfly5207楼2013-09-09 12:26:25
8楼2013-09-17 01:39:06
DBWJ
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10楼2013-10-03 20:12:33