Biology Reference
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Whereas the genome sequence only provides the blueprint of life, the proteome
brings this genome sequence to fruition. Protein expression profiling not only
reveals the overall pattern of protein expression under various environmental
conditions, but also provides information on expression level, post-translational
modifications, and subcellular localization of individual proteins. We are using
functional genomics techniques to focus on two main areas: (1) a basic under-
standing of cell metabolism and stress/starvation responses of S. aureus under
different conditions, and (2) a comprehensive analysis of the function and
structure of virulence-associated regulons.
The first step in the physiological proteomics analysis of S. aureus is the estab-
lishment of a comprehensive two-dimensional (2D) gel proteomemap that should
cover most of the cytoplasmic proteins expressed under specific circumstances
( see Fig. 1 ). For physiological studies, the highly sensitive two-dimensional
polyacrylamide gel electrophoresis (2D PAGE) originally invented by O'Farrel
(2) and Klose (3) is still the state of the art. With our standard 2D gel system
(pH 4-7), we identified 473 proteins that cover about 40% of the cytoplasmic
proteome of S. aureus predicted for this proteomic window (4) . The proteins are
associated with various cellular functions ranging from the transcriptional and
translational machinery, citrate cycle, glycolysis, and fermentation pathways to
biosynthetic pathways of nucleotides, fatty acids, and cell wall components (4,5) .
1st dimension
spot handling platform
Proteome analyzer 4700
2D gel electrophoresis
spots are cut from gel
In-gel digestion
mass spectrometry
Plate: 1 Spot: A25-A4
MS - INTERNAL CALIBRATION
300000
2211.10
250000
MS data
nusA
dnaN
200000
dnaN
atpD
tufA
150000
eno
1045.56
2225.11
N T 01SA0377
100000
p epT
rpoA
eno
1903.92
1875.94
1861.92
1836.94
1797.91
1752.93
50000
2299.18
2255.06
arc pgk
NT01SA0504
1262.60
1259.69
1208.60
1435.75
1327.74
1603.80
2695.24
2667.24
2811.28
2752.28
2724.25
927.63
907.57
1021.58
999.64
1156.56
1106.55
1496.78
1482.78
1940.94
2125.04
2083.02
0
1617.82
2321.13
2839.28
g ap
rpoA
1524.73
1000
1000
1500
1500
2000
2000
2500
2500
3000
3000
3500
3500
Ma ss
Ma ss
MS/MS - PRECURSOR 1047.5715 - INTERNAL CALILIBRATIION
Plate: 1 Spot: A25-A4
1047.64
20000
MS/MS data
15000
10000
1045.65
861.10
839.10
5000
3 2 4.25
270.16
21 1 .15
175.12
508.34
540.35
0
200
300
400
500
600
700
800
900
1000
Mass
mastergel
database alignment
MS/MS data
MS data
Fig. 1. Schematic representation of protein identification by mass spectrometry and
preparation of reference 2D maps.
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