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Proteomics. What is it? How is it done? Are there different kinds? Why would you want to do it (what can it tell you)?. The Central Dogma of Molecular Biology. DNA. transcription. RNA. translation. Protein. Key Concept: Proteins do the work everything else is mostly information. - PowerPoint PPT Presentation
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Proteomics
What is it?
How is it done?
Are there different kinds?
Why would you want to do it (what can it tell you)?
DNA
RNA
Protein
The Central Dogma ofMolecular Biology
transcription
translation
Key Concept: Proteins do the work everything else is mostly information
Key Concept: Proteomics is the “high throughput” analysis of proteins.
DNA Genomics
RNA Genomics/Transcriptomics
Protein Proteomics
Metabolites Metabolomics
The Central Dogma-omics
DNA Genomics ~6-45,000 parts
RNA Genomics/Transcriptomics~100,000 parts
Protein Proteomics~1,000,000 parts
Metabolites Metabolomics>500,000,000 parts
The Central Dogma-omics
Key Concept: Complexity increases the farther away from information you getKey Concept: Chemical complexity also increases the farther away you get
Protein Machines
Key Concept: Biochemical functions are carried out by multi-protein machines
The polyAdenylation Machinery
The Proteosome
Key Concept: A Protein Function can be inferred by it’s binding partners
Key Concept: Knowledge of a Machine’s components is required to understand how it works and how it is regulated
Protein Networks 2 Steps
Key Concept: Protein Machines are organized into larger Networks
Proteins are Organized in Super Networks
Key Concept: The proteome is HIGHLY Networked
Key Concept: Proteins vary widely in concentration
Major Types of ProteomicsInteractomics:
Mapping Protein:Protein Interactions-Yeast 2-hybrid techniques-high throughput protein identification by Mass Spectrometry
Survey Proteomics:Qualitative or Quantitative Analysis of the
protein component-whole organism, tissue, cell type, or subcellular compartment
-2D gel electrophoresis ->MS-typically a few 100
proteins-Multidimensional LC->MS/MS
-typically a few 1000 proteinsIdentification of Biomarkers
Information Trade Offs
Vida infra G. MacBeath
Proteins are Organized in Super Networks
Key Concept: The proteome is HIGHLY Networked
Time of Flight Mass Spectrometer
Key Concept: All Mass Spectrometers have at least 2 parts: an ion source and a mass analyzer
Detectors
Flight tube
Timed ion selectorLaser
Sampleplate
Reflector
Acceleratingfield
++
+
+
+
+ +
Key Concept: Mass Spectrometers can only measure charged species.
A Quadrupole Mass Filter
Ion Trap Mass Spectrometer
Key Concept: Mass Spectrometers can only measure ions and moreaccurately, the behavior of ions in an electric field
A Generic Mass Spectrum
Key Concept: A mass spectrum is a graphical representation of the the data, with intensity on the y axis and m/z on the x axis
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.53
33
17
37
.88
09
12
96
.67
97
96
3.5
27
1
12
10
.68
91
10
21
.55
20
14
25
.62
23
19
01
.88
27
13
53
.60
17
22
42
.16
63
99
5.5
37
5
88
1.2
42
8
10
79
.56
32
12
22
.62
18
17
20
.84
09
15
70
.67
59
112
5.4
92
3
117
4.5
80
4
20
30
.02
36
14
95
.68
21
24
65
.19
26
19
22
.87
02
18
44
.82
45
22
11.0
52
2
25
39
.43
24
1737.49425 1738.56954 1739.64483 1740.72011 1741.79540 1742.87069
Mass (m/z)
1941.2
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
1738.8808
1739.8810
1740.8808
17
37
.88
09
Important Properties of a Mass SpectrumMass is expressed as m/z (mass/charge).
m=1000 z=1 m/z =1001m=1000 z=2 m/z =501m=1000 z=3 m/z =334.3
Intensity is usually normalized to 100Intensity can be expressed as ion current or ion counts
The intensity is related to abundance, but only when you are referring to the same compound.
Mass accuracy is an important parameter that affects downstream analysis. Mass accuracy is usually expressed as ppm. EM=1000.1 TM=1000 ppm= (0.1/1000) x 106
100 ppm
How to Identify Proteins
Key Concept: Each unique protein will give rise to a unique set of peptides
Proteolysis
Proteolysis
Proteolysis
Peptide Mass Fingerprinting
Key Concept: A single protein yields many proteolitic fragments
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.53
33
17
37
.88
09
12
96
.67
97
96
3.5
27
1
12
10
.68
91
10
21
.55
20
14
25
.62
23
19
01
.88
27
13
53
.60
17
22
42
.16
63
99
5.5
37
5
88
1.2
42
8
10
79
.56
32
12
22
.62
18
17
20
.84
09
15
70
.67
59
112
5.4
92
3
117
4.5
80
4
20
30
.02
36
14
95
.68
21
24
65
.19
26
19
22
.87
02
18
44
.82
45
22
11.0
52
2
25
39
.43
24
Proteolysis
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.53
33
17
37
.88
09
12
96
.67
97
96
3.5
27
1
12
10
.68
91
10
21
.55
20
14
25
.62
23
19
01
.88
27
13
53
.60
17
22
42
.16
63
99
5.5
37
5
88
1.2
42
8
10
79
.56
32
12
22
.62
18
17
20
.84
09
15
70
.67
59
112
5.4
92
3
117
4.5
80
4
20
30
.02
36
14
95
.68
21
24
65
.19
26
19
22
.87
02
18
44
.82
45
22
11.0
52
2
25
39
.43
24
Proteolysis
Peptide Mass Fingerprinting
Key Concept: A mass spectrometer is a very accurate scale!
1737.49425 1738.56954 1739.64483 1740.72011 1741.79540 1742.87069
Mass (m/z)
1941.2
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
1738.8808
1739.8810
1740.8808
17
37
.88
09
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% In
te
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.5
33
3
17
37
.8
80
9
12
96
.6
79
7
96
3.5
27
1
12
10
.6
89
1
10
21
.5
52
0
14
25
.6
22
3
19
01
.8
82
7
13
53
.6
01
7
22
42
.1
66
3
99
5.5
37
5
88
1.2
42
8
10
79
.5
63
2
12
22
.6
21
8
17
20
.8
40
9
15
70
.6
75
9
11
25
.4
92
3
11
74
.5
80
4
20
30
.0
23
6
14
95
.6
82
1
24
65
.1
92
6
19
22
.8
70
2
18
44
.8
24
5
22
11
.0
52
2
25
39
.4
32
4
Peptide Mass Fingerprinting
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% In
te
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.5
33
3
17
37
.8
80
9
12
96
.6
79
7
96
3.5
27
1
12
10
.6
89
1
10
21
.5
52
0
14
25
.6
22
3
19
01
.8
82
7
13
53
.6
01
7
22
42
.1
66
3
99
5.5
37
5
88
1.2
42
8
10
79
.5
63
2
12
22
.6
21
8
17
20
.8
40
9
15
70
.6
75
9
11
25
.4
92
3
11
74
.5
80
4
20
30
.0
23
6
14
95
.6
82
1
24
65
.1
92
6
19
22
.8
70
2
18
44
.8
24
5
22
11
.0
52
2
25
39
.4
32
4
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% In
te
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.5
33
3
17
37
.8
80
9
12
96
.6
79
7
96
3.5
27
1
12
10
.6
89
1
10
21
.5
52
0
14
25
.6
22
3
19
01
.8
82
7
13
53
.6
01
7
22
42
.1
66
3
99
5.5
37
5
88
1.2
42
8
10
79
.5
63
2
12
22
.6
21
8
17
20
.8
40
9
15
70
.6
75
9
11
25
.4
92
3
11
74
.5
80
4
20
30
.0
23
6
14
95
.6
82
1
24
65
.1
92
6
19
22
.8
70
2
18
44
.8
24
5
22
11
.0
52
2
25
39
.4
32
4
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078
ENSP00000254108
ENSP00000254636
ENSP00000254719
ENSP00000254942
ENSP00000259848
ENSP00000259925
ENSP00000261366
ENSP00000261700
ENSP00000216019
ENSP00000262570
ENSP00000262584
ENSP00000262637
ENSP00000262709
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% In
te
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.5
33
3
17
37
.8
80
9
12
96
.6
79
7
96
3.5
27
1
12
10
.6
89
1
10
21
.5
52
0
14
25
.6
22
3
19
01
.8
82
7
13
53
.6
01
7
22
42
.1
66
3
99
5.5
37
5
88
1.2
42
8
10
79
.5
63
2
12
22
.6
21
8
17
20
.8
40
9
15
70
.6
75
9
11
25
.4
92
3
11
74
.5
80
4
20
30
.0
23
6
14
95
.6
82
1
24
65
.1
92
6
19
22
.8
70
2
18
44
.8
24
5
22
11
.0
52
2
25
39
.4
32
4
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078
ENSP00000263698
ENSP00000263746
ENSP00000264258
ENSP00000264293
ENSP00000269349
ENSP00000269576
ENSP00000270799
ENSP00000281154
ENSP00000283179
ENSP00000216019
ENSP00000290341
ENSP00000294823
ENSP00000296490
ENSP00000254108
ENSP00000254636
ENSP00000254719
ENSP00000254942
ENSP00000259848
ENSP00000259925
ENSP00000261366
ENSP00000261700
ENSP00000216019
ENSP00000262570
ENSP00000262584
ENSP00000262637
ENSP00000262709
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% In
te
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.5
33
3
17
37
.8
80
9
12
96
.6
79
7
96
3.5
27
1
12
10
.6
89
1
10
21
.5
52
0
14
25
.6
22
3
19
01
.8
82
7
13
53
.6
01
7
22
42
.1
66
3
99
5.5
37
5
88
1.2
42
8
10
79
.5
63
2
12
22
.6
21
8
17
20
.8
40
9
15
70
.6
75
9
11
25
.4
92
3
11
74
.5
80
4
20
30
.0
23
6
14
95
.6
82
1
24
65
.1
92
6
19
22
.8
70
2
18
44
.8
24
5
22
11
.0
52
2
25
39
.4
32
4
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078
ENSP00000263698
ENSP00000263746
ENSP00000264258
ENSP00000264293
ENSP00000269349
ENSP00000269576
ENSP00000270799
ENSP00000281154
ENSP00000283179
ENSP00000216019
ENSP00000290341
ENSP00000294823
ENSP00000296490
ENSP00000254108
ENSP00000254636
ENSP00000254719
ENSP00000254942
ENSP00000259848
ENSP00000259925
ENSP00000261366
ENSP00000261700
ENSP00000216019
ENSP00000262570
ENSP00000262584
ENSP00000262637
ENSP00000262709
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078
ENSP00000233084
ENSP00000233468
ENSP00000240851
Key Concept: Each mass pulls out ~11,000 candidate proteins
Peptide Mass Fingerprinting
ENSP00000031135
ENSP00000084795
ENSP00000198939
ENSP00000201886
ENSP00000202773
ENSP00000216019
ENSP00000216038
ENSP00000216520
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000263698
ENSP00000263746
ENSP00000264258
ENSP00000264293
ENSP00000269349
ENSP00000269576
ENSP00000270799
ENSP00000281154
ENSP00000283179
ENSP00000216019
ENSP00000290341
ENSP00000294823
ENSP00000254108
ENSP00000254636
ENSP00000254719
ENSP00000254942
ENSP00000259848
ENSP00000259925
ENSP00000261366
ENSP00000261700
ENSP00000216019
ENSP00000262570
ENSP00000262584
ENSP00000262637
ENSP00000202773
ENSP00000216055
ENSP00000216038
ENSP00000216019
ENSP00000216832
ENSP00000223129
ENSP00000225430
ENSP00000225792
ENSP00000233078
ENSP00000233084
ENSP00000233468
ENSP00000240851
ENSP00000244357
Key Concept: Ideally only one protein should be uniquely identified
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% In
te
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.5
33
3
17
37
.8
80
9
12
96
.6
79
7
96
3.5
27
1
12
10
.6
89
1
10
21
.5
52
0
14
25
.6
22
3
19
01
.8
82
7
13
53
.6
01
7
22
42
.1
66
3
99
5.5
37
5
88
1.2
42
8
10
79
.5
63
2
12
22
.6
21
8
17
20
.8
40
9
15
70
.6
75
9
11
25
.4
92
3
11
74
.5
80
4
20
30
.0
23
6
14
95
.6
82
1
24
65
.1
92
6
19
22
.8
70
2
18
44
.8
24
5
22
11
.0
52
2
25
39
.4
32
4
Key Concept: Fragments can be matched to a database of proteins.The number of indentified proteins is related to the mass accuracy.
Peptide Mass Fingerprints often FAIL to giveSignificant Results
-Protein Mixtures “confuse” the statistical algorithms-Not enough high quality peaks-Too many “popular masses”-Possible to “play” with the search parameters to give a statistically meaningful result.
Can be overcome by performing peptide fragmentation(AKA MS/MS)
Thermo Demo Movie
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.53
33
17
37
.88
09
12
96
.67
97
96
3.5
27
1
12
10
.68
91
10
21
.55
20
14
25
.62
23
19
01
.88
27
13
53
.60
17
22
42
.16
63
99
5.5
37
5
88
1.2
42
8
10
79
.56
32
12
22
.62
18
17
20
.84
09
15
70
.67
59
112
5.4
92
3
117
4.5
80
4
20
30
.02
36
14
95
.68
21
24
65
.19
26
19
22
.87
02
18
44
.82
45
22
11.0
52
2
25
39
.43
24
Proteolysis
What is MS/MS
-MS/MS is a method in which a peptide is fragmented and the masses of the fragment ions measured.
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.53
33
17
37
.88
09
12
96
.67
97
96
3.5
27
1
12
10
.68
91
10
21
.55
20
14
25
.62
23
19
01
.88
27
13
53
.60
17
22
42
.16
63
99
5.5
37
5
88
1.2
42
8
10
79
.56
32
12
22
.62
18
17
20
.84
09
15
70
.67
59
112
5.4
92
3
117
4.5
80
4
20
30
.02
36
14
95
.68
21
24
65
.19
26
19
22
.87
02
18
44
.82
45
22
11.0
52
2
25
39
.43
24
69 422 775 1128 1481 1834
Mass (m/z)
1449.8
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sit
y
4700 MS/MS Precursor 1737.97 Spec #1 MC[BP = 565.2, 1450]
565.2502
175.1132
353.1470
448.2195 963.41541272.5774
310.2110676.3638
765.3395110.0650 775.4099
648.3871 1585.5645211.1430 399.1859 1062.4656
946.3766530.2659 1695.3250320.1792183.14961563.6715683.3218545.2791 1385.60731174.5771928.4443
757.3596 1555.7285335.1488 475.2506 957.7602631.1696 1226.4336 1513.6215907.8694478.2589
What is MS/MS
-MS/MS is a method in which a peptide is fragmented and the masses of the fragment ions measured.
How do you interpret an MS/MS spectra??
S
AM
P
L
E
R
S
AM
PL
E + R
S
AM
PL E
R
S
AM
P
S
AM
L
E
R
PL
E
R
+
+
+
082405OFRep78 #6779 RT: 36.02 AV: 1 NL: 7.56E6T: ITMS + p ESI Full ms [ 400.00-1700.00]
400 600 800 1000 1200 1400 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
652.45
943.27
627.82 978.09504.73 696.09 787.00 1414.451154.45986.45 1282.55 1515.45 1637.09
082405OFRep78 #6781 RT: 36.03 AV: 1 NL: 8.81E4T: ITMS + c ESI d Full ms2 [email protected] [ 245.00-2000.00]
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
924.57
545.38
767.43
527.33
638.36
881.60
397.25
329.08
1313.061105.82 1474.14
S
AM
PL E
R
+
092304GDH100 #4289-4907 RT: 21.31-25.03 AV: 9 NL: 3.40E3T: Average spectrum MS2 499.73 (4289-4907)
200 300 400 500 600 700 800 900 1000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
813.24
490.26
742.23
542.19184.99
456.14411.21655.26587.17256.05
343.11 688.18 851.26795.30211.05 384.88 634.55 926.34 974.13
How do you interpret an MS/MS spectra??
Steps for Manual Interpretation
1. Label the precursor masses2. Label any obvious water losses
(m/z with 18)3. Look for any peaks with 28 (a – b ion pairs)4. Look up the mass of the first b ion in the dipeptide chart.5. Begin building the b ion series and use the y ion series for confirmation using
the tables.6. Extend till the you reach the presurser mass. (the last amino acid in the b ion series should
be a Lys or Arg and will be +18 relative to themass in the table.)
213.1
99.1
213.1
99.1
AACode MW
AACode MW
AACode MW
AACode MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
99.1
213.1
AACode MW
AACode MW
AACode MW
AACode MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
V
99.1
213.1
AACode MW
AACode MW
AACode MW
AACode MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
NV
114
AACode MW
AACode MW
AACode MW
AACode MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
V N
Q
114 128
AACode MW
AACode MW
AACode MW
AACode MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
I/L
V N
Q
114 128
113
V N
QI/L
GT I/L S E I
K
AACode MW
AACode MW
AACode MW
AACode MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
V N
QI/L
GT I/L S E I
K
AACode MW
AACode MW
AACode MW
AACode MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
VN
QI/L
092304GDH100 #4435-4965 RT: 21.95-25.43 AV: 10 NL: 8.04E3T: Average spectrum MS2 501.27 (4435-4965)
200 300 400 500 600 700 800 900 1000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
770.21
641.21230.96
202.99
437.16
491.51360.07869.25564.09
815.26158.03 385.73290.10 752.23536.14 657.29 879.31 1005.55
175.00 417584.10
826.25
092304GDH100 #4289-4907 RT: 21.31-25.03 AV: 9 NL: 3.40E3T: Average spectrum MS2 499.73 (4289-4907)
200 300 400 500 600 700 800 900 1000
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
ela
tive
Ab
un
da
nce
813.24
490.26
742.23
542.19184.99
456.14411.21655.26587.17256.05
343.11 688.18 851.26795.30211.05 384.88 634.55 926.34 974.13
157.04
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3348.0
0
10
20
30
40
50
60
70
80
90
100
% I
nte
ns
ity
4700 Reflector Spec #1 MC[BP = 863.4, 3348]
86
3.4
27
9
10
59
.53
33
17
37
.88
09
12
96
.67
97
96
3.5
27
1
12
10
.68
91
10
21
.55
20
14
25
.62
23
19
01
.88
27
13
53
.60
17
22
42
.16
63
99
5.5
37
5
88
1.2
42
8
10
79
.56
32
12
22
.62
18
17
20
.84
09
15
70
.67
59
112
5.4
92
3
117
4.5
80
4
20
30
.02
36
14
95
.68
21
24
65
.19
26
19
22
.87
02
18
44
.82
45
22
11.0
52
2
25
39
.43
24
Proteolysis
69 422 775 1128 1481 1834
Mass (m/z)
1449.8
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sit
y
4700 MS/MS Precursor 1737.97 Spec #1 MC[BP = 565.2, 1450]
565.2502
175.1132
353.1470
448.2195 963.41541272.5774
310.2110676.3638
765.3395110.0650 775.4099
648.3871 1585.5645211.1430 399.1859 1062.4656
946.3766530.2659 1695.3250320.1792183.14961563.6715683.3218545.2791 1385.60731174.5771928.4443
757.3596 1555.7285335.1488 475.2506 957.7602631.1696 1226.4336 1513.6215907.8694478.2589
Key Concept: A sample analyzed by MALDI can generate between 10-20 MS/MS spectra before it is consumed.
How to Identify Proteins
Key Concept: A few MS/MS spectra or even MS/MS spectra from a single protein can be manually interpreted
C E Protein Bands are cut from gel,trypsinized and analyzed by massspectrometry
-direct sequencing-search a database (probability based ID)
MS/MS allows for Shot Gun Proteomics
Key Concept: Shot Gun Proteomics lets you look at many things at once
Proteolysis
Proteolysis
Proteolysis
Proteolysis
RT: 0.00 - 89.99
0 10 20 30 40 50 60 70 80Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
34.84
49.29
35.8827.93 39.65
27.75
49.8840.0069.78
18.4854.7625.49
69.3713.5855.30 70.21
7.73 55.81
60.6871.33
6.97 89.20
NL:2.16E7Base Peak F: MS 082405OFRep78
082405OFRep78 #6779 RT: 36.02 AV: 1 NL: 7.56E6T: ITMS + p ESI Full ms [ 400.00-1700.00]
400 600 800 1000 1200 1400 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
652.45
943.27
627.82 978.09504.73 696.09 787.00 1414.451154.45986.45 1282.55 1515.45 1637.09
082405OFRep78 #6781 RT: 36.03 AV: 1 NL: 8.81E4T: ITMS + c ESI d Full ms2 [email protected] [ 245.00-2000.00]
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
924.57
545.38
767.43
527.33
638.36
881.60
397.25
329.08
1313.061105.82 1474.14
Protein ID by Mass Spectrometry
RT: 0.00 - 89.99
0 10 20 30 40 50 60 70 80Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
34.84
49.29
35.8827.93 39.65
27.75
49.8840.0069.78
18.4854.7625.49
69.3713.5855.30 70.21
7.73 55.81
60.6871.33
6.97 89.20
NL:2.16E7Base Peak F: MS 082405OFRep78
082405OFRep78 #6779 RT: 36.02 AV: 1 NL: 7.56E6T: ITMS + p ESI Full ms [ 400.00-1700.00]
400 600 800 1000 1200 1400 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
652.45
943.27
627.82 978.09504.73 696.09 787.00 1414.451154.45986.45 1282.55 1515.45 1637.09
082405OFRep78 #6781 RT: 36.03 AV: 1 NL: 8.81E4T: ITMS + c ESI d Full ms2 [email protected] [ 245.00-2000.00]
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
924.57
545.38
767.43
527.33
638.36
881.60
397.25
329.08
1313.061105.82 1474.14
Protein ID by Mass Spectrometry
~10,000 MS/MS per hourKey Concept: LC-MS/MS data is time restricted by the elution profile of the peptides.Maldi is restricted by sample consumption.
V N
QI/L
GT I/L S E I
K
AACode MW
AACode MW
AACode MW
AACode MW
G 57.02 T 101.04 D 115.02 H 137.05
A 71.037 C 103.01 Q 128.05 F 147.06
S 87.03 L 113.08 K 128.09 R 156.10
P 97.05 I 113.08 E 129.04 Y 163.06
V 99.06 N 114.04 M 131.04 W 186.08
Key Concept: de Novo interpretation is computationally intensive. Most software programs do it by Spectral Matching
acquiredspectrum
Theoreticalspectrum(y/b ions)
100%
0%
1
0
Spectral Matching
acquiredspectrum
theoreticalspectrum(y/b ions)
100%
0%
1
0
x
Spectra matched
Protein ID by Mass Spectrometry
