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yfly520

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[求助] 通过HPLC-MS-MS分析多肽序列

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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comeon,bethebestself

1楼 2013-09-04 02:05:29

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DBWJ

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yfly520: 金币+2, ★★★很有帮助 2013-10-03 20:09:10

这段时间没有上小木虫，没来得及回。
你想得到你蛋白的完整序列是得不到的，因为做的时候应该都用胰蛋白酶处理了，你的蛋白都被打碎了。
这种方法只能间接的得到你蛋白的序列，就是指比对上的蛋白的序列。
而在结果中的peptide就是匹配上的酶解后的序列，也就是你的蛋白中的一部分序列
资料的话还是找找做质谱的公司看看他们能不能提供一些吧，我也没有资料

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9楼2013-09-25 12:33:38

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wizardfan

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感谢参与，应助指数 +1
137167741: 金币+1, 小木虫交流，共同进步~~ 2013-09-17 08:43:17

不知道你到底要的是什么帮助？
LC-MS/MS得到多肽序列常用的有两种，一个是搜数据库：把所有蛋白质都理论上酶解走MS/MS，然后把实际得到的图谱和这些理论的图谱去比对，看哪个更接近，另外一个是de novo根据图谱来推测氨基酸组成。
但是不明白你的结果里有个emPAI, 这个是定量分析，而且这个技术误差很大。

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2楼2013-09-04 06:11:59

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