基于布鲁克道尔顿BioTools软件的一级质谱和二级质谱合并搜库的流程

基于布鲁克道尔顿BioTools软件的一级质谱和二
级质谱合并搜库的流程
1.原理
当蛋白质的酶切峰较少或纯度较低有杂质峰干扰时,单纯依赖一级质谱的PMF检索得分较低,甚至得不到可信的鉴定结果. 而采集肽段的二级质谱并与一级质谱合并后进行检索, 由于合并了一级质谱和二级质谱的信息,可以大大提高蛋白的鉴定得分,提高鉴定的可信度.
2.操作流程
(1)在FlexAnalysis软件中调入相应的一级和二级质谱图,并进行分析和标峰.
(2) 如图1如示, 依次选择一级质谱和二级质谱图,在Tools 菜单中选择BioTools, 逐次将相应的质谱图转入到BioTools软件中.
(3) 如图2如示,在File 菜单中选择Combine TOF + multiple LIFT spectra, 在弹出的对话窗口(如图3)中点出OK,将二级质谱图与对应的一级质谱图进行合并,产生相应的合并文件.
(4)在BioTools软件中,先切换成msms view模式,再点击MS/MS Ion Search按钮(见图4).
检索.
择peptide summary和 protein summary (deprecated)
选项,即可分别得到二级质谱的检索结果和一级二级质谱的合并检索结果.
3. 结果示例
(1)一级质谱单独搜库的结果
Mascot Search Results
User : zcx
Email : chunxi.zhou@
Search title :
Database : Arthrobacter (38729 sequences; 11398127 residues) Timestamp : 23 Jun 2008 at 07:49:37 GMT
Top Score : 44 for gi|116671525, elongation factor Tu [Arthrobacter sp. FB24]
Probability Based Mowse Score
Protein score is -10*Log(P), where P is the probability that the observed match is a random event.
Protein scores greater than 58 are significant (p<0.05).
Concise Protein Summary Report
1. gi|116671525Mass: 43621 Score: 44 Expect: 1.7 Queries
matched: 5
elongation factor Tu [Arthrobacter sp. FB24]
gi|119961333Mass: 43593 Score: 44 Expect: 1.7 Queries matched: 5
elongation factor Tu [Arthrobacter aurescens TC1]
gi|116611634Mass: 43621 Score: 44 Expect: 1.7 Queries matched: 5
translation elongation factor 1A (EF-1A/EF-Tu) [Arthrobacter sp.
FB24]
gi|119948192Mass: 43593 Score: 44 Expect: 1.7 Queries matched: 5
translation elongation factor Tu [Arthrobacter aurescens TC1]
gi|166222696Mass: 43593 Score: 44 Expect: 1.7 Queries matched: 5
Elongation factor Tu (EF-Tu)
gi|166222697Mass: 43621 Score: 44 Expect: 1.7 Queries matched: 5
Elongation factor Tu (EF-Tu)
gi|163840912Mass: 43737 Score: 32 Expect: 27 Queries matched: 4
translation elongation factor Tu [Renibacterium salmoninarum ATCC 33209]
gi|162954388Mass: 43737 Score: 32 Expect: 27 Queries
matched: 4
translation elongation factor Tu [Renibacterium salmoninarum ATCC 33209]
gi|169176286Mass: 18809 Score: 31 Expect: 31 Queries
matched: 3
elongation factor Tu domain protein [Arthrobacter chlorophenolicus A6]
gi|169015530Mass: 18809 Score: 31 Expect: 31 Queries
matched: 3
elongation factor Tu domain protein [Arthrobacter chlorophenolicus A6]
gi|169177251Mass: 4019 Score: 31 Expect: 32 Queries
matched: 2
hypothetical protein AchlDRAFT_3503 [Arthrobacter chlorophenolicus A6]
gi|169014734Mass: 4019 Score: 31 Expect: 32 Queries
matched: 2
hypothetical protein AchlDRAFT_3503 [Arthrobacter chlorophenolicus A6]
Search Parameters
Type of search : Peptide Mass Fingerprint
Enzyme : Trypsin
Fixed modifications : Carbamidomethyl (C)
Variable modifications : Oxidation (M)
Mass values : Monoisotopic
Protein Mass : Unrestricted
Peptide Mass Tolerance : ± 100 ppm
Peptide Charge State : 1+
Max Missed Cleavages : 3
Number of queries : 18
(2) 二级质谱的搜库结果
Mascot Search Results
User : zcx
Email : chunxi.zhou@
Search title :
MS data file : DATA.TXT
Database : Arthrobacter (38729 sequences; 11398127 residues) Timestamp : 18 Jun 2008 at 09:42:28 GMT
Protein hits :
gi|11661
1634
translation elongation factor 1A (EF-1A/EF-Tu)
[Arthrobacter sp. FB24]
gi|162954388
translation elongation factor Tu [Renibacterium salmoninarum ATCC 33209]
Probability Based Mowse Score
Ions score is -10*Log(P), where P is the probability that the observed match is a random
event.
Individual ions scores > 30 indicate identity or extensive homology (p<0.05).
Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.
Peptide Summary Report
Standard scoring MudPIT scoring Show pop-ups
Suppress pop-ups
Require bold red
Error tolerant
1.
gi|116611634 Mass: 43564 Score: 90 Queries matched: 2
translation elongation factor 1A (EF-1A/EF-Tu) [Arthrobacter sp.
FB24]
Check to include this hit in error tolerant search or archive
report
Query Observed Mr(expt)Mr(calc) ppm Mis
s
Scor
e
Expec
t
Ran
k
Peptide
8
1638.892
5
1637.885
2
1637.877
7
4.59
2 48
0.000
87
1
K.VLYDKYPTLNEK
R.D
12
1888.029
9
1887.022
6
1886.985
19.9
1 42
0.004
1
1
R.GITINISHVEYQ
TEKR.H
Proteins matching the same set of peptides:
gi|116671525Mass: 43564 Score: 90 Queries matched: 2
elongation factor Tu [Arthrobacter sp. FB24]
gi|166222697Mass: 43564 Score: 90 Queries matched: 2
Elongation factor Tu (EF-Tu)
2.
gi|162954388Mass: 43680 Score: 90 Queries matched: 2
translation elongation factor Tu [Renibacterium salmoninarum ATCC 33209]
Check to include this hit in error tolerant search or archive
report
Query Observed Mr(expt)Mr(calc) ppm Mis
s
Scor
e
Expec
t
Ran
k
Peptide 8
1638.892
5
1637.885
2
1637.841
3
26.8
1 48
0.000
87
1
K.VLYDKYPTLNEQ
R.D
12
1888.029
9
1887.022
6
1886.985
19.9
1 42
0.004
1
1
R.GITINISHVEYQ
TEKR.H
Proteins matching the same set of peptides:
gi|163840912Mass: 43680 Score: 90 Queries matched: 2
translation elongation factor Tu [Renibacterium salmoninarum ATCC 33209]
match)
Query Observed Mr(expt) Mr(calc) ppm Miss Score Expect Rank Peptid
e
1
845.1151 844.1079
2
861.1085 860.1012
3
877.0880 876.0807
4
1036.55721035.5500
5
1322.63621321.6290
6
1606.78871605.7815
7
1634.93791633.9307
9
1765.77251764.7652
10
1818.92701817.9197
11
1844.93921843.9319
13
2035.99582034.9885
14
2080.21522079.2079
15
2319.13862318.1313
16
2383.98912382.9818
17
2436.38822435.3809
18
2550.20822549.2009
19
2705.22512704.2178
Search Parameters
Type of search : MS/MS Ion Search
Enzyme : Trypsin
Variable modifications : Carbamidomethyl (C),Oxidation (M) Mass values : Monoisotopic
Protein Mass : Unrestricted
Peptide Mass Tolerance : ± 100 ppm
Fragment Mass Tolerance: ± 1.5 Da
Max Missed Cleavages : 3
Instrument type : MALDI-TOF-TOF
(3) 一级质谱和二级质谱的合并搜库结果Mascot Search Results
User : zcx
Email : chunxi.zhou@
Search title :
MS data file : DATA.TXT
Database : NCBInr 20080104 (5824077 sequences; 2008696216 residues)
Timestamp : 18 Jun 2008 at 08:35:43 GMT
Warning : A Peptide summary report will usually give a much clearer picture of MS/MS search results.
Top Score : 138 for gi|163840912, translation elongation factor Tu [Renibacterium salmoninarum ATCC 33209]
Probability Based Mowse Score
Protein score is -10*Log(P), where P is the probability that the observed match is a random event.
Protein scores greater than 80 are significant (p<0.05).
Protein scores are derived from ions scores as a non-probabilistic basis for ranking
protein hits.
Protein Summary Report
Standard scoring MudPIT
scoring
Show pop-ups Suppress pop-ups Require bold red
Index
Accessio n Ma
ss
Sc
ore
Description
1 .
gi|16384
0912
43
737
138
translation elongation factor Tu [Renibacterium
salmoninarum ATCC 33209]
2 .
gi|11667
1525
43
621
90 elongation factor Tu [Arthrobacter sp. FB24]
3 .
gi|11996
1333
43
593
90 elongation factor Tu [Arthrobacter aurescens TC1]
4 .
gi|15297
8672
22
460
70
recombination protein RecR [Actinobacillus
succinogenes 130Z]
Results List
1. gi|163840912Mass: 43737 Score: 138 Expect: 9.2e-
008 Queries matched: 5
translation elongation factor Tu [Renibacterium salmoninarum ATCC 33209]
Observed Mr(expt)Mr(calc) ppm Star
t End Mis
s
Ion
s
Peptide
1638.892
5 1637.885
2
1637.841
3
26.8
35
-
47 1
48
K.VLYDKYPTLNEQR.D
1844.939
2 1843.931
9
1843.888
7
23.4
267
-282
--- K.QLDEAWAGENCGLLLR
.G
1888.029
9 1887.022
6
1886.985
19.9
62
-
77 1
42 R.GITINISHVEYQTEKR
.H
2035.995
8 2034.988
5
2034.948
9
19.5
322
-336
--- R.HNPFYSNYRPQFYFR.
T
2550.208
2 2549.200
9
2549.162
5
15.1
317
-336
1
--- K.DEGGRHNPFYSNYRPQ
FYFR.T
No match to: 845.1151, 861.1085, 877.0880, 1036.5572, 1322.6362,
1606.7887, 1634.9379, 1765.7725, 1818.9270, 2080.2152, 2319.1386, 2383.9891, 2436.3882, 2705.2251
2. gi|116671525Mass: 43621 Score: 90 Expect: 0.0052 Queri
es matched: 5
elongation factor Tu [Arthrobacter sp. FB24]
Observed Mr(expt)Mr(calc) ppm Star
t End Mis
s
Ion
s
Peptide
1818.927
0 1817.919
7
1817.879
6
22.1
158
-174
--- R.ELLSSQGFDGDEAPVV
R.V
1844.939
2 1843.931
9
1843.888
7
23.4
267
-282
--- K.QLDEAWAGENCGLLLR
.G
1888.029
9 1887.022
6
1886.985
19.9
62
-
77 1
42 R.GITINISHVEYQTEKR
.H
2035.995
8 2034.988
5
2034.948
9
19.5
322
-336
--- R.HNPFYSNYRPQFYFR.
T
2550.208
2 2549.200
9
2549.162
5
15.1
317
-336
1
--- K.DEGGRHNPFYSNYRPQ
FYFR.T
No match to: 845.1151, 861.1085, 877.0880, 1036.5572, 1322.6362,
1606.7887, 1634.9379, 1638.8925, 1765.7725, 2080.2152, 2319.1386, 2383.9891, 2436.3882, 2705.2251
3. gi|119961333Mass: 43593 Score: 90 Expect: 0.0052 Queri
es matched: 5
elongation factor Tu [Arthrobacter aurescens TC1]
Observed Mr(expt)Mr(calc) ppm Star
t End Mis
s
Ion
s
Peptide
1818.927
0 1817.919
7
1817.879
6
22.1
158
-174
--- R.ELLSSQGFDGDEAPVV
R.V
1844.939
2 1843.931
9
1843.888
7
23.4
267
-282
--- K.QLDEAWAGENCGLLLR
.G
1888.029
9 1887.022
6
1886.985
19.9
62
-
77 1
42 R.GITINISHVEYQTEKR
.H
2035.995
8 2034.988
5
2034.948
9
19.5
322
-336
--- R.HNPFYSNYRPQFYFR.
T
2550.208
2 2549.200
9
2549.162
5
15.1
317
-336
1
--- K.DEGGRHNPFYSNYRPQ
FYFR.T
No match to: 845.1151, 861.1085, 877.0880, 1036.5572, 1322.6362,
1606.7887, 1634.9379, 1638.8925, 1765.7725, 2080.2152, 2319.1386, 2383.9891, 2436.3882, 2705.2251
4. gi|152978672Mass: 22460 Score: 70 Expect: 0.52 Queries
matched: 4
recombination protein RecR [Actinobacillus succinogenes 130Z]
Observe
d Mr(expt
)
Mr(calc
)
ppm Star
t
En
d
Mis
s
Ion
s
Peptide
1634.93
79 1633.93
07
1633.87
87
31.8
2
-15
---
M.QSSPLLENLVEHLR.C
1638.89
25 1637.88
52
1637.78
65
60.3
38
-53
1
--- R.SGGMNLARALTDAMSK.I +
Oxidation (M)
1765.77
25 1764.76
52
1764.91
92
-
87.2
5
1
-15
---
-.MQSSPLLENLVEHLR.C
1888.02
99 1887.02
26
1886.97
13
27.2
105
-12
1
25 R.YFVLMGHLSPLDGIGPR.E +
Oxidation (M)
No match to: 845.1151, 861.1085, 877.0880, 1036.5572, 1322.6362,
1606.7887, 1818.9270, 1844.9392, 2035.9958, 2080.2152, 2319.1386, 2383.9891, 2436.3882, 2550.2082, 2705.2251
Search Parameters
Type of search : MS/MS Ion Search
Enzyme : Trypsin
Fixed modifications : Carbamidomethyl (C)
Variable modifications : Oxidation (M)
Mass values : Monoisotopic
Protein Mass : Unrestricted
Peptide Mass Tolerance : ± 100 ppm
Fragment Mass Tolerance: ± 1.5 Da
Max Missed Cleavages : 1
Instrument type : MALDI-TOF-TOF
Query1 (845.1151,1+) : <no title>
Query2 (861.1085,1+) : <no title>
Query3 (877.0880,1+) : <no title>
Query4 (1036.5572,1+) : <no title>
Query5 (1322.6362,1+) : <no title>
Query6 (1606.7887,1+) : <no title>
Query7 (1634.9379,1+) : <no title>
Query8 (1638.8925,1+) : <no title>
Query9 (1765.7725,1+) : <no title>
Query10 (1818.9270,1+) : <no title>
Query11 (1844.9392,1+) : <no title>
Query12 (1888.0299,1+) : <no title>
Query13 (2035.9958,1+) : <no title>
Query14 (2080.2152,1+) : <no title>
Query15 (2319.1386,1+) : <no title>
Query16 (2383.9891,1+) : <no title>
Query17 (2436.3882,1+) : <no title>
Query18 (2550.2082,1+) : <no title>
4. 讨论
本例是银染一维分离蛋白胶点的分析.由于样品量少,二级谱图的质量不太好,单纯二级质谱数据的检索得分与不高,但与一级质谱数据合并后检索得分大大得高,使原本PMF检索没有可信结果的数据得到可信的检索结果.。