protein identification by maldi mass spectrometry karin hjernø and peter roepstorff department of...
TRANSCRIPT
Protein Identification by
MALDI Mass Spectrometry
Karin Hjernø and Peter Roepstorff Department of Biochemistry and Molecular Biology
University of Southern Denmark
Denmark
Peptide Mass Fingerprint (PMF)2) Enzymatic digestion (trypsin)
3) Micropurification ( salt, + concentration)
4) Mass spectrometric analysis, data interpretation.
1) Separation of proteins, here by 2D-gels
pI
Mw
TheoreticalDigestion
Digestion
Protein of Interest
MALDI-MS
...LIHGFYMNKPL...
...LVCDERTFGHG...
...HYIGFREWMKL...
...LIYTSARDEFW...
.................
Databaseof known
Sequences
Experimental Peptide Masses
TheoreticalPeptide Masses
Comparison
679.52 684.34 842.50 856.52 864.48 870.531045.571126.571314.771794.87...
1074.501087.561151.531194.601200.631240.621246.571275.631323.681353.73...
Protein ID
PMF
Enzymes for PMF
www.matrixscience.com
Trypsin•High specificity•Peptides in a mass range compatible with MALDI•Small enzyme
MALDI-MS spectrum
m/z956.0 966.0
Inte
nsi
ty3.50
0
958.46
C:\Documents and Settings\Administrator\Desktop\yeast\b21735al\735calred.massml (10:50 10/29/01)
Description: NoName NoName
m/z2702.0 2711.0
Inte
nsi
ty
2.50
0
2705.2
C:\Documents and Settings\Administrator\Desktop\yeast\b21735al\735calred.massml (11:36 10/29/01)
Description: NoName NoName
1 Da1 Da
842.5 1602.8 2720.2
Three examples of a normal isotopic distribution at different m/z-value.
m/z 838.0 850.0
Intensity
40
0
m/z 1598.0 1612.0
Intensity
1.00
0
m/z 2716.0 2730.0
Intensity
1.80
0
m/z1276.0 1286.0
Inte
nsi
ty
18
0
1277.7 1279.7
C:\Documents and Settings\Administrator\Desktop\backupdisks\Karin\Andrea\Spot # 9341 16.5.00 b14856al.massml (11:27 01/15/02)
Description: Yeast
m/z1852.0 1860.0
Inte
nsi
ty
6.00
0
1852.0
C:\Documents and Settings\Administrator\Desktop\backupdisks\Karin\Andrea\Spot # 8538 16.5.00 b14934al.massml (11:29 01/15/02)
Description: Yeast
?
Isotope distribution
Removal of contaminants
Common contaminants•Keratin•Tryptic autodigest projects (842.5, 2211.1)
•Matrix-clusters
Overlapping matrix (871.94) and peptide (873.50) peaks.
m/z 500 4000
Inte
nsity
60
0
973.
43
1179
.5
Ker
atin
1 K
erat
in1
Ker
atin
1 Ker
atin
9
Ker
atin
9 K
erat
in1
Ker
atin
1 Ke
ratin
1 K
erat
in9
Ker
atin
9 K
erat
in1
Ker
atin
1
Ker
atin
9
Ker
atin
1
C:\Documents and Settings\Administrator\Desktop\keratinrigtspectra.massml (10:52 01/15/02) Description: Yeast
Removal of contaminantsPage selection
Read peak list from clipboard
Copy peak list to clipboard
Perform multipoint calibration
Show/hide graph window
Set window for erazor list
Select erazor list
Select sections in erazor list
Display options
Project name
List information
Mass info
Mass list
Peak Erazor
www.welcome.to/GPMAW
m/z1000 5000
Inte
nsity
25
0
1638
.71475
.6
842.
42
2704
.82210
.8
1851
.7
1993
.7
1267
.6
2500
.91307
.5
2202
.9
1765
.5
3338
.333
24.3
3146
.2
2932
.1
1179
.5
2716
.7
2383
.6
2185
.9
<33
55.2
>
1707
.6
2234
.922
24.8
2398
.7
1794
.6
1657
.617
59.7
1280
.6
2283
.9
2149
.8
2444
.9
1715
.6 1837
.7
830.
37
1818
.7
2342
.7
1320
.4
2509
.8
1234
.5
2183
.8
1384
.6
1251
.6
1434
.6
2565
.9
2790
.128
22.0
2748
.126
44.0
1323
.5
2583
.925
89.9
1503
.615
23.6
2807
.0
1036
.4
870.
44
2901
.2
3819
.6
3662
.3
1045
.5
2872
.0
1082
.5
3050
.9
875.
36
3677
.4\\Bmb-filsrv\PRpublic\IngerMZ\PH0508\p54731ic, plug 3.massml (11:21 03/15/05)Description: PH0508, Ag-plug 3
Outlier
Taking advantage of contaminants!
Identification of contaminants
Multipoint calibration on contaminants
< 20 ppm
What!? ... No calibrants..
m/z1000 5000
Inte
nsity
60
0
3905
.9
2568
.5
976.
28
2321
.4
2459
.4
4166
.72851
.5
1592
.3
1336
.3
2550
.5
1576
.3
2865
.5
4182
.6
2582
.5
2305
.4
951.
30
4150
.7
1318
.3 2587
.6
2430
.4
1954
.4
1047
.3
849.
24
1593
.3
2216
.522
90.5
2200
.5
1993
.4
2353
.423
39.4
2704
.4
1953
.4
4294
.6
1608
.3
2525
.5
2103
.521
18.5
2716
.3
1475
.4
1620
.3
1307
.4
1963
.4
1765
.3
1978
.4
919.
25
1794
.3
4032
.7
2601
.6
842.
31
1356
.4
4310
.6
1447
.414
05.3
1277
.4
1106
.3
1658
.4
1063
.3
1464
.4
935.
2599
2.24
820.
23
2444
.4
\\Bmb-filsrv\PRpublic\IngerMZ\PH0507\p54545ic, gelplug 5.massml (11:39 03/15/05)Description: PH0507, gelplug 5
The mass defect: Difference between the monoisotopic and the integer mass value of a given amino acid residue,
The mass defect of a peptide around 8-900 Da is .5, around 2000 Da it is .0 (mass dependent)
< 50 ppm
Filteret and calibrated peak list, what then?!?
Make a database dependent search;
•Open the prefered search program
•Choose the search database
•Choose the search parameters
•Start the search....... Protein candidates
Mascot Interface
927.4719960.5380999.61301026.57271055.53721066.56411072.57671088.60551099.54881129.66791199.67241211.6559
.......
.......
.......
.......1768.94411782.91711867.84181894.96841924.99951982.08342056.98542142.05942310.22162720.3082
Results List
1. gi|6321412 Mass: 76977 Score: 103 tryptophan synthetase; Trp5p [Saccharomyces cerevisiae]Observed Mr(expt) Mr(calc) Delta Start End Miss Peptide 759.43 758.42 758.41 0.01 695 - 700 0 IGWDLR 830.45 829.44 829.44 -0.00 439 - 445 0 QALNVFR 1 Pyro-glu (N-term Q)847.47 846.47 846.47 -0.01 439 - 445 0 QALNVFR 907.51 906.50 906.50 0.00 473 - 479 0 FWVTNLK1003.53 1002.52 1002.57 -0.05 438 - 445 1 RQALNVFR1031.46 1030.45 1030.48 -0.02 273 - 280 0 DEFFAFQK1267.72 1266.71 1266.77 -0.06 385 - 396 0 INNALAQVLLAK
1319.64 1318.63 1318.69 -0.05 207 - 219 0 DTPLAVGFGVSTR1458.70 1457.69 1457.76 -0.07 269 - 280 1 VLSKDEFFAFQK
1484.77 1483.76 1483.79 -0.02 677 - 690 1 GDKDVQSVAEVLPK1583.75 1582.74 1582.79 -0.05 361 - 373 0 LTEHCQGAQIWLK 1607.80 1606.79 1606.85 -0.05 344 - 357 0 SLYSYIGRPSSLHK1664.86 1663.85 1663.89 -0.05 620 - 635 0 AQFIAATDAQALLGFK1702.76 1701.76 1701.79 -0.03 423 - 437 0 FGLTCTVFMGAEDVR
………...
Non-significant matches
1 MSEQLRQTFA NAKKENRNAL VTFMTAGYPT VKDTVPILKG FQDGGVDIIE 51 LGMPFSDPIA DGPTIQLSNT VALQNGVTLP QTLEMVSQAR NEGVTVPIIL 101 MGYYNPILNY GEERFIQDAA KAGANGFIIV DLPPEEALKV RNYINDNGLS 151 LIPLVAPSTT DERLELLSHI ADSFVYVVSR MGTTGVQSSV ASDLDELISR 201 VRKYTKDTPL AVGFGVSTRE HFQSVGSVAD GVVIGSKIVT LCGDAPEGKR 251 YDVAKEYVQG ILNGAKHKVL SKDEFFAFQK ESLKSANVKK EILDEFDENH 301 KHPIRFGDFG GQYVPEALHA CLRELEKGFD EAVADPTFWE DFKSLYSYIG 351 RPSSLHKAER LTEHCQGAQI WLKREDLNHT GSHKINNALA QVLLAKRLGK 401 KNVIAETGAG QHGVATATAC AKFGLTCTVF MGAEDVRRQA LNVFRMRILG 451 AKVIAVTNGT KTLRDATSEA FRFWVTNLKT TYYVVGSAIG PHPYPTLVRT 501 FQSVIGKETK EQFAAMNNGK LPDAVVACVG GGSNSTGMFS PFEHDTSVKL 551 LGVEAGGDGV DTKFHSATLT AGRPGVFHGV KTYVLQDSDG QVHDTHSVSA 601 GLDYPGVGPE LAYWKSTGRA QFIAATDAQA LLGFKLLSQL EGIIPALESS 651 HAVYGACELA KTMKPDQHLV INISGRGDKD VQSVAEVLPK LGPKIGWDLR 701 FEEDPSA
Sequence Coverage: 51%
Significant matchesp < 0.05
m/z500 4000
Inte
nsity
18
0
1891.9
<3810.9><3340.7>
2037.02191.1
1319.6
1882.01475.7
1708.9
2066.0
1307.6
2211.1
1994.0
1393.7
1702.8 1948.0
1435.71179.5
2284.2
1607.81458.7
1277.7
1638.8 2403.22705.2
842.50
1234.61073.6
2717.12249.1
1045.5958.46
2754.4
823.56
847.47759.43
2807.3578.47
1 MSEQLRQTFA NAKKENRNAL VTFMTAGYPT VKDTVPILKG FQDGGVDIIE 51 LGMPFSDPIA DGPTIQLSNT VALQNGVTLP QTLEMVSQAR NEGVTVPIIL 101 MGYYNPILNY GEERFIQDAA KAGANGFIIV DLPPEEALKV RNYINDNGLS 151 LIPLVAPSTT DERLELLSHI ADSFVYVVSR MGTTGVQSSV ASDLDELISR 201 VRKYTKDTPL AVGFGVSTRE HFQSVGSVAD GVVIGSKIVT LCGDAPEGKR 251 YDVAKEYVQG ILNGAKHKVL SKDEFFAFQK ESLKSANVKK EILDEFDENH 301 KHPIRFGDFG GQYVPEALHA CLRELEKGFD EAVADPTFWE DFKSLYSYIG 351 RPSSLHKAER LTEHCQGAQI WLKREDLNHT GSHKINNALA QVLLAKRLGK 401 KNVIAETGAG QHGVATATAC AKFGLTCTVF MGAEDVRRQA LNVFRMRILG 451 AKVIAVTNGT KTLRDATSEA FRFWVTNLKT TYYVVGSAIG PHPYPTLVRT 501 FQSVIGKETK EQFAAMNNGK LPDAVVACVG GGSNSTGMFS PFEHDTSVKL 551 LGVEAGGDGV DTKFHSATLT AGRPGVFHGV KTYVLQDSDG QVHDTHSVSA 601 GLDYPGVGPE LAYWKSTGRA QFIAATDAQA LLGFKLLSQL EGIIPALESS 651 HAVYGACELA KTMKPDQHLV INISGRGDKD VQSVAEVLPK LGPKIGWDLR 701 FEEDPSA
Tryptic auto digest
Contaminants
1 MSEQLRQTFA NAKKENRNAL VTFMTAGYPT VKDTVPILKG FQDGGVDIIE 51 LGMPFSDPIA DGPTIQLSNT VALQNGVTLP QTLEMVSQAR NEGVTVPIIL 101 MGYYNPILNY GEERFIQDAA KAGANGFIIV DLPPEEALKV RNYINDNGLS 151 LIPLVAPSTT DERLELLSHI ADSFVYVVSR MGTTGVQSSV ASDLDELISR 201 VRKYTKDTPL AVGFGVSTRE HFQSVGSVAD GVVIGSKIVT LCGDAPEGKR 251 YDVAKEYVQG ILNGAKHKVL SKDEFFAFQK ESLKSANVKK EILDEFDENH 301 KHPIRFGDFG GQYVPEALHA CLRELEKGFD EAVADPTFWE DFKSLYSYIG 351 RPSSLHKAER LTEHCQGAQI WLKREDLNHT GSHKINNALA QVLLAKRLGK 401 KNVIAETGAG QHGVATATAC AKFGLTCTVF MGAEDVRRQA LNVFRMRILG 451 AKVIAVTNGT KTLRDATSEA FRFWVTNLKT TYYVVGSAIG PHPYPTLVRT 501 FQSVIGKETK EQFAAMNNGK LPDAVVACVG GGSNSTGMFS PFEHDTSVKL 551 LGVEAGGDGV DTKFHSATLT AGRPGVFHGV KTYVLQDSDG QVHDTHSVSA 601 GLDYPGVGPE LAYWKSTGRA QFIAATDAQA LLGFKLLSQL EGIIPALESS 651 HAVYGACELA KTMKPDQHLV INISGRGDKD VQSVAEVLPK LGPKIGWDLR 701 FEEDPSA
~5500 Da
•Maldi mass range : from ~ 700 Da to ~ 3500
•Suppression effect (preferential ionization of some components at the expence of others)
•Post-translational modifications
Why not 100% sequence coverage
Example – a classical example
Manual evaluation – what to look for?
•Likely/unlikely missed cleavage sites
•Overlapping peptides
•Partial modifications
•Mass accuracy
•(Intensity of the peaks)
•……
N CR R RPKR K
N C
N
Missed cleavage sites
Digestion using trypsin
Missed cleavages•Only missed cleavages of one of the following kind are highlylikely to be observed (relative to other sites): R/KxxxxxxxR/K
xxxxxxxR/KR/KxR/KxxxxxxR/KxxxxE/D R/KxxxxR/KxxxxE/Dx R/KxxxxR/KxxxxR/K E/DxxxxR/KxxxxR/K xE/DxxxxR/KxxxxR/K PxxxxR/K
Basic or acidic residue close to the cleavage site in question
Digestion using trypsin
N CR R RPKR K
N C
N
RxE
DR
KR
Manual evaluation
•Likely/unlikely missed cleavage sites
•Overlapping peptides
•Partial modifications
•Mass accuracy
•(Intensity of the peaks)
•……
Digestion using trypsin
N CR R RPKR K
N C
NDR
KR
M
Mox
Inte
nsity
121
811.
782
(179
4.8
17)
177
9.7
93
178
3.8
00
182
7.7
86
m/z1780 18300
0
113
6.5
m/z1250 1300
Inte
nsity
70
0
128
2.6
38
125
0.6
61
125
4.6
44
126
6.6
41
129
8.6
33
+4
+16 +32
+48
Non-modified
HVFSATQLAAM(ox)FIDK
m/z 1680.0 1700.0
Intensity
16
0
1678.8
1694.8
C:\Documents and Settings\Administrator\Desktop\backupdisks\Karin\Andrea\yeast\b21738al\738calnotred.massml (13:44 01/15/02) Description: NoName NoName
16 Da
HVFSATQLAAMFIDK
m/z830.0 850.0
Inte
nsity
6.00
0
847.47
Tryptic autodigest
QALNVFR
Pyro-QALNVFR
830.45
17 Da
Partial modification
Oxidized tryptophan (W)
Oxidized methionine N-terminal pyro-glutamate
Manual evaluation – what to look for?
•Likely/unlikely missed cleavage sites
•Overlapping peptides
•Partial modifications
•Mass accuracy
•(Intensity of the peaks)
•……
Example – a classical example
Example 2 – a false-positive
m/z800 2200
Inte
nsi
ty
30
0
20
60
.9
19
14
.01
90
1.0
16
77
.9
15
82
.8
14
95
.9
12
87
.6
12
18
.7
10
92
.6
10
18
.5
87
0.5
6
10
47
.6
10
90
.61
12
8.6
11
45
.61
15
1.6
12
17
.71
25
9.6
12
63
.81
29
8.7
14
15
.7 14
39
.81
46
3.8
14
69
.81
45
6.8
15
43
.9
17
79
.8
21
28
.0
22
11
.10
0
10
45
.55
7
12
99
.80
7
22
25
.10
9
84
2.5
09
6
22
83
.13
8
17
94
.84
4
11
46
.62
5
22
33
.10
4
13
09
.67
7
22
98
.16
5
82
4.5
24
3
Spot # 241 8.2.00 b12420al.massml
Example 2 – a false-positive
Example 2 – a false-positive
842.5096927.4719960.5380999.61301026.57271045.57271055.53721066.56411072.57671080.58851088.60551099.54881129.66791197.66231199.67241211.6559
.......
.......
.......
.......1768.94411782.91711867.84181894.96841904.03021924.99951982.08342045.04592056.98542142.05942211.09962310.22162720.30822807.2930
Peak List
Protein mixtures
m/z500 2500
Inte
nsi
ty
25
0
13
61
.76
15
92
.99
12
90
.69
14
46
.78
12
42
.68
16
94
.88
14
50
.74
20
56
.99
16
39
.95
15
67
.77
13
46
.68
14
87
.74
14
67
.87
16
23
.82
12
53
.65
17
68
.94
16
21
.78
12
11
.66
10
88
.61
17
17
.91
11
97
.66
17
82
.92
11
29
.67
19
25
.00
16
87
.87
22
11
.1010
55
.54
10
99
.55
10
45
.57
21
42
.06
18
94
.97
10
72
.58
84
2.5
10
10
80
.59
27
20
.31
20
45
.05
19
04
.03
28
07
.29
96
0.5
38
99
9.6
13
92
7.4
72
23
10
.22
10
26
.57
10
66
.56
11
99
.67
14
39
.80 14
79
.82
17
21
.91
17
38
.90
18
67
.84
19
82
.08
\\Hermes\prgroup\Karin\Andrea!\Spot # IT 2023 20.9.01 b23753al.massml (11:20 11/02/01)Description: Human
Annexin VI
Trypsin autodigest
Protein mixtures
Protein mixtures
m/z500 2500
Inte
nsi
ty
25
0
13
61
.76
15
92
.99
12
90
.69
14
46
.78
12
42
.68
16
94
.88
14
50
.74
20
56
.99
16
39
.95
15
67
.77
13
46
.68
14
87
.74
14
67
.87
16
23
.82
12
53
.65
17
68
.94
16
21
.78
12
11
.66
10
88
.61
17
17
.91
11
97
.66
17
82
.92
11
29
.67
19
25
.00
16
87
.87
22
11
.1010
55
.54
10
99
.55
10
45
.57
21
42
.06
18
94
.97
10
72
.58
84
2.5
10
10
80
.59
27
20
.31
20
45
.05
19
04
.03
28
07
.29
96
0.5
38
99
9.6
13
92
7.4
72
23
10
.22
10
26
.57
10
66
.56
11
99
.67
14
39
.80 14
79
.82
17
21
.91
17
38
.90
18
67
.84
19
82
.08
\\Hermes\prgroup\Karin\Andrea!\Spot # IT 2023 20.9.01 b23753al.massml (11:20 11/02/01)Description: Human
Annexin VI
Hypothetical protein XP_038637(heat shock 70D)
Trypsin autodigest
Protein mixtures
D11m/z1000 3500
Inte
nsi
ty
140
0
19
06
.98
4
14
67
.81
5
20
77
.08
0
22
92
.14
8
16
07
.83
4
13
02
.70
8
17
16
.85
3
14
34
.79
3
11
75
.65
31
12
7.5
62
28
73
.37
72
90
3.4
13
When MS/MS is needed...
When MS/MS is needed...
When MS/MS is needed...
Digestion
Protein of Interest
MALDI-MS
(MALDI-) MS/MS
RAWGYVLE
Protein ID
MS and MS/MS
Verification of protein ID, analysis of unassigned peaks
Tandem mass spectrometer
Mass analyzer
Ion Source DetectorMass
analyzerCC
Precursor selection
Collision Cell, fragmentation
of ionsSeparationof fragments
MS of a peptide mixture
899.013
MS/MS of a peptide2+
(collision energy 10 eV)899.013
MS/MS of a Peptide (collision energy 15 eV)
899.013
MS/MS of a Peptide(collision energy 18 eV)
899.013
Peptide fragmentation
Roepstorff and Fohlman, 1984
Biemann, 1988
Paizs and Suhai, 2004
Formation of b- and y-ions
Identification of the peptide LLQVVEEPQALAAFLR
Y1Y2
Y3Y4
Y5
Y6
Y7Y8
Y10
Y11
Y12
Y13
Y9899.013
Y13E E V V
Jens Andersen, Odense, Denmark
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
Manuel Interpretation
115
Amino Acid 3 LetterCodeSingle Letter
Code Residue Mass
Monoisotopic
Glycine Gly G 57.02147
Alanine Ala A 71.03712
Serine Ser S 87.03203
Proline Pro P 97.05277
Valine Val V 99.06842
Threonine Thr T 101.04768
Cysteine Cys C 103.00919
Isoleucine Ile I 113.08407
Leucine Leu L 113.08407
Asparagine Asn N 114.04293
Aspartic Acid Asp D 115.02695
Glutamine Gln Q 128.05858
Lysine Lys K 128.09497
Glutamic Acid Glu E 129.04260
Methionine Met M 131.04049
Histidine His H 137.05891
Phenylalanine Phe F 147.06842
Arginine Arg R 156.10112
Tyrosine Tyr Y 163.06333
Tryptophan Try W 186.07932
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
D
Manuel Interpretation
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
D
128146
Manuel Interpretation
Amino Acid 3 LetterCodeSingle Letter
Code Residue Mass
Monoisotopic
Glycine Gly G 57.02147
Alanine Ala A 71.03712
Serine Ser S 87.03203
Proline Pro P 97.05277
Valine Val V 99.06842
Threonine Thr T 101.04768
Cysteine Cys C 103.00919
Isoleucine Ile I 113.08407
Leucine Leu L 113.08407
Asparagine Asn N 114.04293
Aspartic Acid Asp D 115.02695
Glutamine Gln Q 128.05858
Lysine Lys K 128.09497
Glutamic Acid Glu E 129.04260
Methionine Met M 131.04049
Histidine His H 137.05891
Phenylalanine Phe F 147.06842
Arginine Arg R 156.10112
Tyrosine Tyr Y 163.06333
Tryptophan Try W 186.07932
146??
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
D
Q/K
Manuel Interpretation
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
D
Q/K
172
Manuel Interpretation
Amino Acid 3 LetterCodeSingle Letter
Code Residue Mass
Monoisotopic
Glycine Gly G 57.02147
Alanine Ala A 71.03712
Serine Ser S 87.03203
Proline Pro P 97.05277
Valine Val V 99.06842
Threonine Thr T 101.04768
Cysteine Cys C 103.00919
Isoleucine Ile I 113.08407
Leucine Leu L 113.08407
Asparagine Asn N 114.04293
Aspartic Acid Asp D 115.02695
Glutamine Gln Q 128.05858
Lysine Lys K 128.09497
Glutamic Acid Glu E 129.04260
Methionine Met M 131.04049
Histidine His H 137.05891
Phenylalanine Phe F 147.06842
Arginine Arg R 156.10112
Tyrosine Tyr Y 163.06333
Tryptophan Try W 186.07932
172?!?
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
D
Q/K
172
57
Manuel Interpretation
Amino Acid 3 LetterCodeSingle Letter
Code Residue Mass
Monoisotopic
Glycine Gly G 57.02147
Alanine Ala A 71.03712
Serine Ser S 87.03203
Proline Pro P 97.05277
Valine Val V 99.06842
Threonine Thr T 101.04768
Cysteine Cys C 103.00919
Isoleucine Ile I 113.08407
Leucine Leu L 113.08407
Asparagine Asn N 114.04293
Aspartic Acid Asp D 115.02695
Glutamine Gln Q 128.05858
Lysine Lys K 128.09497
Glutamic Acid Glu E 129.04260
Methionine Met M 131.04049
Histidine His H 137.05891
Phenylalanine Phe F 147.06842
Arginine Arg R 156.10112
Tyrosine Tyr Y 163.06333
Tryptophan Try W 186.07932
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
D
Q/KG
115
Manuel Interpretation
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
D
Q/KG
D
Manuel Interpretation
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
F
DQ/K
GD
DL
Manuel Interpretation
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
F
DQ/K
GD
DL
Manuel Interpretation
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
GDG
Manuel Interpretation
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
Immonium ions, ”diagnostic” ions
72 83 94 105 116 127
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
84.0678
102.0426
74.0513101.0969
72.0 100.2 128.4 156.6 184.8 213.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Int
ensit
y
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.0683
84.0678 129.0862
173.0822
102.0426
74.0513147.0961 191.0951101.0969 183.0528 203.0798175.0881159.0696
K/Q
I/L
T
F
K/Q
E
Immonium ions, ”diagnostic” ions
69.0 444.6 820.2 1195.8 1571.4 1947.0
Mass (m/z)
2.9E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
4700 MS/MS Precursor 1844.93 Spec #1 MC=>NF0.7[BP = 120.1, 29008]
120.0661
86.0839
201.06831035.360284.0678 792.2761
1225.3296
348.1187 907.2828417.1365
1053.3120620.2520173.0822 435.13651150.3596
235.08091297.4041
102.0426 330.11821497.4641
288.0954 1716.5573530.161474.0513 1410.4763735.2722 1827.02581110.3387401.1511 1597.5017191.0951 574.1970303.1168 1282.3550836.1899 949.2646 1468.4775
1. Ion series: DGD(Q/K)DFL
2. Ion series: GDG
Immonium ions: F,T,(I/L),E,(Q/K)
Proline-induced fragmenation (N-terminal to Proline; Xaa|Pro)
Most abundant when the Xaa is Val, His, Ile, Leu, Asp
Not abundant when Xaa is Pro or GlyBreci, 2003; Kapp, 2003
Fragmentation of VVAASLNPVDFK, singly charged ion
Fragmentation of VPTVDVSVVDLTVR , singly charged ion
No y13???
Peptide with proton predominantly associated with basic residue
The proton is mobilized through by collisional activation
NH2
NN
N
O
R1
R2
OR3
O
N
R4
OB
CO2H
H+
NH2
NN
N
O
R1
R2
OR3
O
N
R4
OLys
CO2HH+
Proton mobility
If an arginine is present (most basic residue), then the proton is sequestered (non-mobile) and require more energy for mobilization
NH2
NN
N
O
R1
R2
OR3
O
N
R4
OArg
CO2H
H+
CID or PSD
NH2
NN
N
O
R1
R2
OR3
O
N
R4
OArg
CO2H
H+
H+
If more protons than arginines are present, then a mobile proton will be present
MALDI ESI
Acidic-induced fragmentation
Paizs and Suhai
Arg
Asp
Also Glu, but to a lesser degree
Charge-remote fragmentation in contrast to Charge-induced fragmentation
Fragmentation of VPTVDVSVVDLTVR , singly charged ion
y4y9
Dominating fragmentation-pathways for singly charged MALDI-peptide ions
LSMOXTNDPLEAAR
Y6 Y3 Y1
64
64
Y6
Y3Y1
Verification of MALDI-TOF/TOF search result
Peptides containing ArgParent mass Intensity parent mass Sequence Modification Expected MS/MS ions
1479.15 37417 LGEYGFQNALIVR 175.12 300.16 1180.651566.86 138278 DAFLGSFLYEYSR 175.12 116.03 425.21 1143.54 1452.72928.45 78882 YLYEIAR 175.12 359.24 569.26
1193.86 170361 DTHKSEIAHR missed cleavage 175.12 116.03 496.30 698.31 1078.58712.97 12683 SEIAHR 175.12 217.08 496.30
1477.16 20315 FwGKYLYEIAR 2-W_Ox2/ 175.12 359.24 1119.51 1313.651084.16 18261 YLYEIARR missed cleavage 175.12 569.26 515.341053.21 13897 CCTKPESER 175.12 175.12 391.19 662.26 878.341283.69 14464 HPEYAVSVLLR 175.12 364.16 920.561439.31 264249 RHPEYAVSVLLR missed cleavage 175.12 520.26 920.56
Peptides containing LysParent mass Intensity parent mass Sequence Modification Expected MS/MS ions
1566.86 138278 LKPDPNTLcDEFK 9-C_Cysteic_acid/ 242.19 454.27 1114.48 1326.561249.70 22359 FKDLGEEHFK missed cleavage1163.95 1448 LVNELTEFAK1143.01 1510 KQTALVELLK missed cleavage886.56 46360 DDSPDLPK 244.17 318.09 569.33 643.26
Prediction of intense ions
The human proteomeIsoelectric point (pI)
Molecular weight (Mw)
Marked spots are differentially expressed between normal red strawberries and a white mutant (half up-regulated and half down-regulated).
(fragaria ananassa)
Strawberry (not sequenced)
Hjernoe et al, 2005, Proteomics, in press
Difference in protein expression visual by eye
Difference in protein expression detected by the differential analysis software (here DeCyder)
Red contra white strawberries
Number of spots
Proteins found to be down regulated
Protein identified based on homology to
1 Flavanone 3- hydroxylase Onobrychis viciifolia
1 Dihydroflavonol reductase Arabidopsis thaliana
4 O-methyltransferase Fragaria x ananassa
4 Chalcone synthase Fragaria x ananassa
.... .... ....
All four proteins are known to be involved in the flavonoid biosynthesis pathway. One of the functions of flavonoids is to act as a pigment, giving colour to fruits.
MS/MS
MS
Search combining both MS and MS/MS spectra
Example of protein identification based on MS/MS spectra from a MALDI-TOF/TOF instrument
Search against all green plants
Significant hit
Details from the search result
4-sulfophenyl-isothiocyanate (SPITC)
Marakov et al, J. Mass Spectrom. (2003) 38; 373-377
Wang et al., Rapid.Commun. Mass. Spectrom (2004), 18(1); 96-102
SPITC-derivatization, an N-terminal Sulfonation
The peptide needs a net-charge of +1 in order to be detected
Derivatization of peptides
m/z500 2000
Inte
nsi
ty
10.00
0 + SPITC
- SPITC
R E AG I T I V Q G D P L E Y N
MS/MS Fragmentation of NYELPDGQVITIGAERFound in gi|21538, actin [Solanum tuberosum]
215 Da
1774
1774
1989
Karin Hjernø
Fragaria x ananassa
MALDI-TOF/TOF spectra obtained at Applied Biosystems 4700 Proteomics Analyzer
b2 b3
y1
y1
y2y3
y4
y5
y6
y7
y8 y9
y10
y11
y12
y13y14
y15
y10
y12
y13
898
920 1051
963
903 905
880
825834
418433 455 465 478 487
664 663 708 715
745
780 784
96
118 157161
212213277
281
297 301324325 334 349
352396
397404409
416
1112
Chalcone synthase
O-methyltransferase
Dihydroflavonol reductaseFlavanone 3-hydroxylase
Red
919
1026
899
294
499 509
The Bet v 1-homologous strawberry allergen, Fra a 1
898
9201051
919
898
9201051
919
777
Red
White
Mw
pI
Strawberry allergen-containing spots
K* L L T G G H P A S V L SPITC
K* L V T G H P A S V L SPITC
K* L V T G G H P A S V L SPITC
m/z200 1400
Inte
nsi
ty
01392
1435
1449
One spot, three distinct isoforms
The End....