Database
Searches
Peptide mass fingerprinting
digest
Spectrum
processing
MS
Protein
(gel band or spot)
Mass spectrum
peptides
Protein X theoretical digest
1554.25
2055.39
1942.44
1755.67
987.55
855.34
677.68
Peak list
Search
Protein Y theoretical digest
Report
HIT
Protein X
Protein Y
Protein Z
SCORE
1000
50
5
Protein Z theoretical digest
Data processing …before
searching
• Baseline correct
• Noise reduction
• Peak deisotope for monoisotopic peaks
…what is a monoisotopic peak
• Copy to Excel and sort for peak
intensity
• Search with the top 75 peak ions
(peptides)
Generate mass list of all
peptides
• Computer program used to convert
graph data (from mass spectrum) to
table data (list of peptide masses)
V
o
y
a
g
e
r
S
p
e
c
#
1
[
B
P
=
8
4
2
.
5
,
2
2
2
0
7
]
1045.6139
100
7 2 8 2 .1
972.5954
1007.467
973.4202
1023.462
973.4202
1036.481
975.6104
1066.493
977.4627
1082.488
982.4603
1105.601
983.4991
1153.524
998.4552
1172.581
1473.8500
90
80
2212.1514
% Intensity
70
1155.6985
60
50
1494.7859
40
1524.8172
1908.0468
1179.6516
30
1107.5970
20
1003.5450
10
1000.0361
0
1000
1666.8767
1310.7056
1608.9940
1324.7029
1788.0128
1477.8171
1210.6405
1718.8852
1215.0833 1425.9303
1600
2054.0554
2300.2157
2226.1625
2104.1794
2081.0745
2692.3379
2385.0063
2313.1521
2550.2102
2200
2788.4984
2800
M ass (m/z )
3024.5809
3294.0001
3400
3603.6864
0
4000
Intensity
Simple Mass Spectrum
771.9
1165.2
361.4
607.7
320.3
992.1
681.7
200
400
600
800
1000
Mass (Da)
1200
1400
1600
320.3
361.4
607.7
681.7
771.9
992.1
1165.2
List of masses
Database search using
computer algorithm
e.g. MASCOT
EAT
EGR
ISPYK
EMETR
EMANYK
PLEASEMAK
EATSTHEYAR
Sequence matches
PLEASEMAKEMANYKRISPYKREMETREATSTHEYAREGREAT
Mass list of all peptides in the
mass spectrum copied into here
www.matrixscience.com
Coverage = 26%
We found peptides that
make up only ¼ of the
entire protein – but this
is enough for a
confident identification!
QUESTIONS