Transcript A microwave and detergent procedure to detect high

A microwave and detergent procedure to detect high molecular mass proteins from
vegetative bacteria by MALDI-TOF MS
Elizabeth Patton1; Nathan Edwards2; Berk Oktem3; and Catherine Fenselau1
Chemistry and Biochemistry and 2Center for Bioinformatics, University of Maryland, College Park, MD; 3 Middle Atlantic Mass Spectrometry Lab, Johns Hopkins School of Medicine, Baltimore, MD
%Int.
%Int.
90
Escherichia coli
80
Escherichia coli
70
35205
57335
55165
56159
59496
Salmonella typhimurium
80
50510
20
90
52106
35540
100
47873
40
%Int.
45793
46624
60000
43285
44097
55000
41287
50000
37389
38275
39147
45000
35418
40000
Mass/Charge
32388
33416
34421
35000
30348
31191
30000
28514
25000
25905
26665
20000
24654
60
0
20226
21198
22140
22991
59496
56159
80
57335
55165
52106
50510
47873
45793
10
46624
44097
41287
100
42682
38275
37389
39147
43285
35614
34421
32388
33416
30348
28514
20
31191
25905
26665
24654
25617
24227
30
Disrupt cell membrane
and solubilize proteins with
acid cleavable detergent
Intact Cells
20887
40
22313
20226
50
22991
35418
60
0
20000
25000
30000
35000
40000
45000
50000
55000
60000
Mass/Charge
70
60
35751
50
45533
36867
37925
43289
35950
34664
32578
33487
31161
28424
10
29755
24658
26687
Zwitterionic 6-PPS Detergent [2]
25920
20
22325
20247
30
23010
40
0
20000
25000
30000
35000
40000
45000
50000
55000
60000
Mass/Charge
20703
58888
59760
0.7
50653
47797
49199
46479
10
O
0.8
43542
42840
44286
39786
38200
37525
20
36862
36156
34153
35252
46640
33710
0.9
32883
27725
+
31212
30
29023
40
25764
24250
27404
32682
22418
50
N
54713
41336
22760
60
Bacillus subtilis 168
1
30468
O
70
24917
O
80
23026
O-
90
21741
S
H3C
100
20212
O
Clean up protein with a
Folch extraction [3]
Mix protein with saturated
sinapinic acid (SA)
Lower pH and microwave
%Int.
20032
CH3
0
20000
25000
30000
35000
40000
45000
50000
55000
60000
21116
%Int.
100
21905
Mass/Charge
CH3
90
80
C22H39NO5S
24008
44113
30226
26624
25517
24693
20
10
0
0
20000
25000
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40000
45000
50000
55000
Comparison of Comet Macromizer spectra of four species
The cryodetector is more sensitive to higher masses
because the signal is independent of mass and, thus,
impact velocity.
43334
37515
38049
38420
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17714
15553
Escherichia coli
Salmonella typhimurium
Salmonella typhimurium
91406
77979
Salts
103682
100kD
50918
43243
20kDa
protein fraction at the
interface
 Lipids
Bacillus subtilis 168
Bacillus anthracis Sterne
23972
21063
21785
127445
89082
83965
43000
36082
163864
126474
Search Rapid Microorganism
Identification DataBase
for best match
112667
116000
CHCl3
3
5
10
8
peaks
Solvent System [3]
H2O
60000
Mass/Charge
MALDI-TOF instruments:
Kratos Axima CFR+ and
Comet Macromizer
CH3OH
50
30
20
ppm
15
0.1
36273
20293
21628
30
22972
21317
0.2
35493
•
40
37670
•
Enhances the solubility of
hydrophobic proteins
Acid cleavable by microwaving at
pH 1.4
Eliminates detergent signal
interference
50
100838
•
0.3
60
Sample applied on top
of dried layer of saturated
SA crystals on slide
0.6
chance
m atch
0.5
probability
0.4
Bacillus anthracis Sterne
70
1. To evaluate accessibility by MALDI to
higher mass proteins in intact bacteria
2. To evaluate the suitability of higher
mass proteins to provide identifications
based on genomic database searching
1. Fenselau, C.; Demirev, P. A. Characterization
of intact microorganisms by MALDI mass
spectrometry. Mass Spectrom. Rev. 2001, 20,
157-171.
2. Norris, J. L.; Porter, N. A.; Caprioli, R. M. Mass
spectrometry of intracellular and membrane
proteins using cleavable
detergents.
Analytical Chemistry 2003, 75, 6642-6647.
3. Wessel, D.; Flugge, U. I. A Method for the
Quantitative Recovery of Protein in DiluteSolution in the Presence of Detergents and
Lipids. Analytical Biochemistry 1984, 138,
141-143.
Spectra are reproducible from spot to spot.
Comparison of Axima spectra of four species
100
Objectives
Literature cited
Reproducibility
Results
54793
Recent research has proposed rapid and
robust
identification
of
intact
microorganisms using matrix assisted laser
desorption/ ionization time-of-flight mass
spectrometry
and
bioinformatics
[1].
Previous work has relied primarily on
desorption and detection of protein
biomarkers weighing less than 20 kilo
Daltons. Analysis of prokaryotic genomes
predicts fewer proteins with higher masses
per organism and, thus, the potential to
provide more definitive microorganism
identifications. However, higher mass
proteins have not yet been readily
accessible by MALDI and widely evaluated
for rapid detection of bacteria. They are
difficult to desorb because of suppression
by other components of the lysed cell; they
are detected with less sensitivity by most
commercial ion detectors. This poster
reports a procedure using acid-cleavable
detergent and microwave to facilitate
desorption of higher molecular weight
protein biomarkers from lysed whole cells.
This approach has been evaluated with
Escherichia
coli
(K-12),
Salmonella
typhimurium, Bacillus anthracis Sterne, and
Bacillus subtilis (168).
Experimental methods and
materials
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Introduction
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10
13
5
15
20
This simulation illustrates that high-mass, in the
20-50KDa
range,
is
insufficient
for
microorganism identification with current
technologies.
Ribosomal proteins from the
RMIDB database were selected and then a
random error, based on the instrument accuracy
(x-axis), was applied to their exact theoretical
masses; the RMIDB was used to "look-up" the
spectrum (number of peaks detected on y-axis)
and check the e-value (z-axis) of the correct
answer.
Conclusion
This procedure afforded the desorption and
detection of several proteins from intact species in
molecular mass ranges above 20kDa. However,
good spectra with peaks in the 20-50kDa range
aren't enough for microorganism identification. To
make high-mass biomarkers suitable requires one
or more of the following advances, all of which
increase the specificity of a spectrum peak with
respect to its species/organism.
a) a significant boost in mass accuracy,
b) an understanding of which proteins, or protein
subset, have abundant peaks in these spectra,
c) obtaining peaks in a mass range above 80kDa.