Anal. Chem.

1999, 71, 3226-3230

Identification of Bacterial Proteins Observed in

MALDI TOF Mass Spectra from Whole Cells
Ricky D. Holland, Christopher R. Duffy, Fatemeh Rafii, John B. Sutherland, Thomas M. Heinze,
Claude L. Holder, Kent J. Voorhees, and Jackson O. Lay Jr*,

Divisions of Chemistry and Microbiology, Food and Drug Administration, National Center for Toxicological Research,
Jefferson, Arkansas 72079, and Department of Chemistry, Colorado School of Mines, Golden, Colorado 80401

Characteristic ions in the MALDI TOF mass spectra from

bacterial cells have been associated with four known
proteins. The proteins, observed both from cells and in
filtered cellular suspensions, were isolated by HPLC and
identified on the basis of their mass spectra and their
partial amino acid sequence, determined using the
Edman method (10-15 residues). The acid resistance
proteins HdeA and HdeB give rise to ions near m/z 9735
and 9060 in MALDI TOF mass spectra from cells and
from extracts of both Escherichia coli 1090 and
Shigella flexneri PHS-1059. However, the proteins
associated with proteolytic cleavage by the peptidase Lep,
rather than the precursor proteins, were observed, both
using cells and from cellular extracts. A cold-shock
protein, CspA, was associated with the ion near m/z 7643
from Pseudomonas aeruginosa. Similarly, a cold-acclimation protein, CapB, was identified as the source of
the ion near m/z 7684 in P. putida. This last protein
was homologous with a known CapB from P. fragi. While
these experiments involved the detection of known or
homologous proteins from typical bacteria, this same
approach could also be applied to the detection of unique
proteins or biomarker proteins associated with other
bacteria of public health significance.
Chemotaxonomy represents one approach to the characterization of bacteria. With this method, classification is based upon
the measurement of chemical constituents, either as unique
components (often called biomarkers) or as contributors to distinct
ratios of common chemical components. Chemotaxonomy can be
used to differentiate species of bacteria or to differentiate strains
of related organisms. In principle, chemotaxonomy using mass
spectrometric, rather than chromatographic, or other timeintensive methods, could shorten analysis times enough to justify
the greater expense of the mass spectrometry (MS) instrumentation. Use of MS for the characterization of bacteria was investigated as early as 1975 by Anhalt and Fenselau.1 In a series of
studies, Fenselau and co-workers evaluated fast atom bombard* To whom correspondence should be addressed. Tel.: (870) 543-7288. Fax:
(870) 543-7686. E-mail: jlaycnctr.fda.gov.
Division of Chemistry, Food and Drug Administration.
Division of Microbiology, Food and Drug Administration.
Colorado School of Mines.
(1) Anhalt, J. P.; Fenselau, C. Anal. Chem. 1975, 47, 219-225.

3226 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

ment, laser desorption, and plasma desorption mass spectrometry

for the analysis of bacteria.1-5 In these early studies, the principal
analytes were phospholipids or other small-molecule biomarkers
for bacteria. The use of sample pyrolysis with pattern recognition
has been the basis for the identification of intact bacteria using
mass spectrometry.6 Chemotaxonomy has also been successfully
demonstrated using GC/MS techniques. For example, Fox et al.
have demonstrated the use of GC/MS for differentiation of two
closely related pathogenic organisms, Bacillus anthracis and
B. cereus,7 that are difficult to differentiate phenotypically or
genotypically.
In 1994, Cain et al.8 reported the use of MALDI TOF/MS to
differentiate bacteria on the basis of the analysis of proteins
isolated from disrupted cells. Proteins were isolated from crude
cellular extracts with methanol. This work was novel because the
bacterial biomarkers detected were proteins rather than the
smaller molecules examined in the earlier pyrolysis or laser
desorption studies. The analysis of bacterial cells, rather than
extracted proteins, by MALDI/TOF MS can be viewed as a logical
extension of prior experiments. Holland et al.9 successfully
characterized bacteria, rather than extracts, on the basis of ions
formed by MALDI directly from bacterial cells using conditions
much like those used for the analysis of isolated proteins. MALDI/
TOF MS gave signals in excess of 10 000 daltons from cellular
components that were presumed to be proteins. The successful
identification of a small test set of blind-coded bacteria9 and the
appearance, within a few months, of similar results in two other
laboratories10,11 suggested that MALDI/TOF MS might be a useful
tool for the characterization of bacteria. Subsequently, this
(2) Heller, D. N.; Fenselau, C.; Cotter, R. J.; Demirev, P.; Olthoff, J. K.; Honovich,
J.; Uy, M.; Tanaky, T.; Kishimoto, Y. Biochem. Biophys. Res. Commun. 1987,
142, 194-199.
(3) Ho, B. C.; Fenselau, C.; Hansen, G.; Larsen, J.; Daniel, A. A. Clin. Chem.
1983, 29, 1349-1353.
(4) Fenselau, C.; Cotter, R. J. Chem. Rev. 1987, 87, 501.
(5) Heller, D. N.; Cotter, R. J.; Fenselau, C.; Uy, O. M. Anal. Chem. 1987, 59,
2806-2809.
(6) DeLuca, S.; Sarver, E. W.; Harrington, P. D.; Voorhees, K. J. Anal. Chem.
1990, 62, 1465-1472.
(7) Fox, A.; Rogers, J. C.; Fox, K. F.; Schnitzer, G.; Morgan, S. L.; Brown, A.;
Aono, R. J. Clin. Microbiol. 1990, 546-552.
(8) Cain, T. C.; Lubman, D. M.; Weber, W. J., Jr. Rapid Commun. Mass Spectrom.
1994, 8, 1026-1031.
(9) Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. E.;
Voorhees, K. J.; Lay, J. O., Jr. Rapid Commun. Mass Spectrom. 1996, 10,
1227-1232.
(10) Claydon, M. A.; Davey, S. N.; Edward-Jones, V.; Gordon, D. B. Nature
Biotechnol. 1996, 14, 1584-1586.
10.1021/ac990175v CCC: $18.00

1999 American Chemical Society

Published on Web 06/16/1999

technique has been used to distinguish 25 different strains of

Escherichia coli.12
Although many of the same ions were present upon reanalysis
of bacterial cultures, the ratios of ion intensity values and the
specific ions observed for bacteria almost always varied somewhat
in replicate experiments.9-11 Part of this variation could be
described as method-related differences in spectra obtained from
otherwise identical bacteria. An interlaboratory comparison by
researchers at the University of Alberta and at the U. S. Armys
Aberdeen Proving Ground facilities has demonstrated that methodrelated variation can be controlled.13 Unfortunately, another major
source of variation in spectra from bacteria is associated with
differences in the biology of the bacteria which is based on
environmental conditions, and this is potentially much more
difficult to control. For bacteria obtained from the environment
rather than laboratory settings, variability in the MALDI TOF mass
spectra, associated with the organisms response to the environment, may be much more problematic than method-related
variation, precisely because of the ability of MS to detect such
small changes in chemical composition. Characterization of the
protein biomarkers in the MALDI spectra of bacteria may lead to
a better understanding of these biology-based changes, and the
identification of specific biomarker ions should facilitate the use
of specific ions for chemotaxonomic purposes.
In this work, we demonstrate the isolation and characterization
of several proteins responsible for characteristic ions in the
MALDI/TOF MS spectra from bacteria. Some of the preliminary
work was reported in ref 14.
EXPERIMENTAL SECTION
Preparation of samples. The bacteria Shigella flexneri PHS1059, E. coli 1090, Pseudomonas aeruginosa, and P. putida were
grown on tryptic soy agar for 24 h. Colonies were suspended in
1 mL of 2:1 double distilled water/acetonitrile (J. T. Baker,
Phillipsburg, NJ) with 0.1% trifluoroacetic acid (Fluka Chemical,
Ronkonkoma, NY) in 1.5 mL polypropylene microcentrifuge tubes
to make a very cloudy suspension. The bacterial suspension was
then centrifuged using a 5415C Eppendorf centrifuge (Eppendorf,
Madison, WI) set at 600g for 2-3 min. Supernatants from
S. flexneri and E. coli were then filtered (0.5-m Millipore filter)
and stored at 5 C until HPLC analysis. The filtered bacterial
extracts from P. aeruginosa and P. putida were vacuum evaporated
to dryness and redissolved in 200 L of 2:1 water/acetonitrile with
0.1% TFA prior to HPLC separation.
Isolation and sequencing of proteins. The bacterial components from cell extracts were separated and isolated by HPLC
using a 4.6 250 mm Vydac C18 protein/peptide column (Vydac,
Hesperia, CA). About 100 L of the bacterial extract (filtered
suspension) was used for each injection onto the HPLC column.
The mobile phases were water and acetonitrile with 0.1% TFA in
both phases. The gradient was 20-90% acetonitrile (linear) over
(11) Krishnamurthy, T.; Ross, P. L. Rapid Commun. Mass Spectrom. 1996, 10,
1992-1996.
(12) Arnold, R. J.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1998, 12, 630636.
(13) Wang, Z.; Russon, L.; Li, L.; Roser, D. C.; Long, S. R. Rapid Commun. Mass
Spectrom. 1998, 12, 456-464.
(14) Holland, R. D.; Rafii, F.; Holder, C. L.; Heinze, T. M.; Sutherland, J. B.;
Voorhees, K. J.; Lay, J. O., Jr. Proceedings of the 46th ASMS Conference on
Mass Spectrometry and Allied Topics, Orlando, FL, 1998; p 154.

30 min, held at 90% for 5 min, then back to 20% after 3 min. The
flow rate (Varian 9012 HPLC pump, Varian Instrument Group,
Sugarland, TX) was 1.0 mL/min. Components in the suspension
were detected at 260 nm with a Varian 9050 detector. All major
HPLC separated components were manually collected in 1.5-mL
microcentrifuge tubes. The solutions containing components were
vacuum-evaporated to dryness using a Savant SCA110A system
(Savant Instruments, Inc., Holbrook, NY). The system was set at
65 C. The dried components were dissolved in 10 L of 0.1%
aqueous TFA for analysis. Specific HPLC separated components
containing the proteins of interest were identified on the basis of
MALDI/TOF MS analysis of each.
Each of the components showing a response for m/z 9060,
and 9735 from S. flexneri were collected 20 times, and the HPLC
separated components corresponding to the same mass were
pooled. Similarly, an HPLC separated component containing m/z
9739 from E. coli was collected 20 times and pooled. For the
P. putida and P. aeruginosa, HPLC separated components containing peaks at m/z 7643 and 7684, respectively, were collected.
However, for these two bacteria, only two injections were made,
from more concentrated samples. All these purified and pooled
HPLC separated components were sent to Midwest Analytical,
Inc., (St. Louis, MO) for automated Edman analysis. Proteins were
identified using the BLAST program from the National Center
for Biotechnology Information.15,16
MALDI TOF/MS Analysis. The matrix (R-cyano-4-hydroxycinnamic acid) (Sigma Chemical Co., St. Louis, MO) was dissolved
in 2:1 water:acetonitrile with 0.1% TFA. The matrix was added
beyond the saturation point, shaken vigorously, and centrifuged
for 1 to 2 min at 14 000 rpm. The clear liquid was decanted for
use. Fresh matrix was prepared daily. Bovine cytochrome c or
myoglobin from horse heart (Sigma) were used as standards for
instrument calibration or as internal mass standards. The calibration standards (1 mg/mL) were prepared in 0.1% TFA in water.
The HPLC fractions and filtered bacterial extracts were analyzed
using 2 L from a 9:2 (L each, matrix/sample) mixture.
Unfiltered bacterial suspensions were analyzed using a 18:2 ratio.
The calibration standard was prepared at a 27:2 matrix/analyte
ratio mixture. For each matrix/analyte mixture, 2 L was placed
on separate 2-mm sample pins and allowed to air-dry. The samples
were placed in a Vestec model YM200 (Vestec, Inc., Houston,
TX) linear time-of-flight mass spectrometer. The samples were
desorbed with a Nd:YAG laser with the frequency tripled at 355
nm. The acceleration voltage was set at 26 000 V. Detector
response was monitored using a Tektronix TDS 520 digitizing
oscilloscope (Beaverton, OR). Mass assignments were made using
a commercial software package (Grams 386, Galactic, Inc.,
Salem, NH).
RESULTS AND DISCUSSION
Acid-Resistance Proteins. The first step in the identification
of the proteins giving rise to signals in the MALDI/TOF mass
spectra was their detection in cellular suspensions. Prominent ions
were observed near m/z 9060 and 9735 in MALDI/TOF mass
spectra from cells of S. flexneri, using the method described in
(15) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. J. Mol.
Biol. 1990, 215, 403-410.
(16) Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J.; Zhang, Z.; Miller,
W.; Lipman, D. J. Nucleic Acids Res. 1997, 25, 3389-3402.

Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

3227

Figure 1. MALDI/TOF mass spectra of the supernatants from

Shigella flexneri and Escherichia coli. (See text for conditions.)

Figure 3. MALDI/TOF mass spectra of isolated proteins 1 and 2

from Shigella flexneri. (See text for conditions.)

Figure 2. HPLC separation of proteins 1 and 2 from Shigella

flexneri. (See text for conditions.)

ref 9 (data not shown). These same ions were also observed, using
cells, with a closely related bacteria, E. coli. These two ions were
detected in cellular supernatants from both bacteria (see Figure
1). The mass assignments for both components (m/z 9060 and
9735) showed a measured standard deviation of (5 daltons on
the basis of triplicate analyses. (The precision in these mass
measurements is typical of our results with other bacteria and
proteins using this instrument.) Figure 2 shows an HPLC
chromatogram from the same supernatant of a centrifuged
suspension of S. flexneri. MALDI/TOF MS analyses of individual
HPLC fractions were used to determine the retention times for
target proteins. They were found in fractions corresponding to
the peaks marked 1 and 2 in Figure 2, and their MALDI/TOF
mass spectra are shown in Figure 3. Mass spectral analysis of
the other major HPLC peaks associated with the separation (data
not shown) revealed that many other proteins also observed in
the MALDI spectrum from bacteria were present in the supernatant as well, but these have not yet been identified.
For protein identification, peaks 1 and 2 were separated by
HPLC, and the corresponding peaks were pooled. The two pooled
samples were purified in a final HPLC step (Figure 4) and
submitted to automated Edman analysis. On the basis of the first
10 amino acid residues from each protein, the identities were
determined to be homologous with hns deletion-induced protein
3228 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

Figure 4. HPLC trace from the pooled samples of proteins 1 and

2 from Shigella flexneri. (See text for conditions.)

B (HdeB) (m/z 9060) and hns deletion-induced protein A (HdeA)

(m/z 9735) from E. coli.17 Both of these proteins are associated
with acid resistance.20 The first 10 N-terminal amino acids
sequenced by the Edman method, for the HPLC component
labeled 1 in Figure 2, were ANESAKDMTC, corresponding to a
portion (residues 34-43) of the sequence for the HdeB precursor
from E. coli strain K12. Figure 5a shows the complete sequence
for E coli K12 protein HdeB where the bold portion (residues 34112) of the sequence depicts the smaller portion of a homologous
protein actually collected from S. flexneri. The predicted mass for
this sequence is 9064 daltons (Table 1), in good agreement with
the measured mass of 9060 daltons (see Table 1, Figure 3b).
Similarly, for the HPLC separated component corresponding to
peak 2 in Figure 2, the first 10 amino acids sequenced by the
Edman method were ADAQKAADNK, corresponding to a portion
(residues 22-31) of the sequence for the HdeA precursor from
(17) Link, A. J.; Robison, K.; Church, G. M. Electrophoresis 1997, 18, 12591313.
(18) Yoshida, T.; Ueguchi, C.; Mizuno, T. J. Bacteriol. 1993, 175, 7747-7748.
(19) Arnqvist, A.; Olsen, A.; Normark, S. Mol. Microbiol. 1994, 13, 1021-1032.
(20) Yoshida, T.; Ueguchi, C.; Yamada, H.; Mizuno, T. Mol. Gen. Genet. 1993,
237, 113-122.

Figure 6. Amino acid sequence of (a) P. aeruginosa CspA and (b)

P. fragi CapB.
Figure 5. Amino acid sequence of E. coli K12 (a) HdeB and (b)
HdeA.
Table 1: Proteins Identified from Bacterial Extracts
bacterium

protein

HPLC
tr (min)

observed
massa

expected
massb

mass
differencec

S. flexneri
S. flexneri
E. coli
P. aeruginosa
P. putida

HdeA
HdeB
HdeA
CspA
CapBd

10.9
10.5
10.9
7.3
8.2

9735
9060
9739
7643
7684

9739
9064
9739
7605

-4
-4
0
38

a From suspended cells, in daltons. b For the protonated molecule,

predicted from the sequences in Figures 5-8, in daltons. c Difference
between the expected and observed masses, in daltons. d Homologue
of P. fragi CapB; the actual mass for the P. putida protein is unknown.

E. coli. The expected mass, based on the protein sequence shown

in bold (residues 22-110, Figure 5b), is 9739, in good agreement
with 9735 daltons, the observed mass (Figure 3a).
The segments of proteins identified in our study are both part
of larger precursor proteins. They are consistent with the expected
proteolytic cleavage by the signal peptidase, Lep, between residues
33 and 34 for HdeB and residues 21 and 22 for HdeA.17 These
proteins are encoded by genes on the hns-dependent expression
AB operon (hdeAB),18 which is regulated by the stationary-phasespecific sigma factor (s).19 Although the function of the two
proteins is not known,18,20 they are associated with the acidresistance phenotype21 which allows bacteria to survive acidic
conditions in the stomach.
Both of these proteins isolated from S. flexneri were homologous to proteins associated with E. coli K12. This is consistent
with phylogenetic studies reporting that these bacteria might even
be classified as a single species.22 Nevertheless, to provide
additional evidence that these two components were actually
homologous proteins, one of them was also characterized from
E. coli. The protein believed to be HdeA (identical to peak 2 from
S. flexneri) was isolated and characterized from E. coli exactly as
described above. Analysis of the corresponding E. coli HPLC
separated component (peak 2) by Edman degradation gave the
same amino acid sequence observed with S. flexneri. Moreover,
the mass determined by MALDI was 9739 daltons (Table 1), in
perfect agreement with the predicted nominal mass. (An HPLC
separated component (m/z 9060, peak 1) with the same retention
behavior as HdeB has also been isolated from E. coli but not
(21) Waterman, S. R.; Small, P. L. C. Mol. Microbiology 1996, 21, 925-940.
(22) Wang, R. F.; Cao, W. W.; Cerniglia, C. E. Mol. Cell. Probes 1997, 11, 427432.

sequenced.) Thus, the HdeA protein gives rise to the same signal
in the MALDI/TOF MS spectra of E. coli and S. flexneri using
either cells or extracts, and this evidence, as well as the
phylogenetic studies cited above, strongly suggests that the same
is true for the HdeB protein.
Interestingly, the acid resistance proteins reported in this work
and in our preliminary report14 were not identified in a recent study
using a peptide mapping approach, even though they were
detected.23 Our work suggests two possible reasons why these
proteins might not have been identified by peptide mapping. First,
the proteins might have been resistant to proteolysis. In our
laboratory, we have been unable to produce good fragments from
HdeA or HdeB using trypsin (data not shown). Second, as noted
above, proteolytic cleavage by Lep resulted in smaller proteins
than were expected from the genome database. Deviations from
the masses or sequences predicted on the basis of the genome
may confound attempts to identify proteins exclusively on the basis
of mass spectrometry measurements, unless such changes are
accounted for in some way. For example, a study of the E. coli
proteome indicated that 60% of the proteins encoded in the
genome were proteolytically processed.17 In this study, the Edman
method was critical to the identification of these proteins.
Cold-Induced Proteins. On the basis of the observation that
MALDI/TOF mass spectra of bacteria change upon storage at 5
C,24 the presence of cold shock proteins was anticipated. A protein
isolated from P. aeruginosa, with a mass of 7643 daltons, was
collected in the same manner as the proteins described above
(Table 1). This same mass was observed both from cells and
HPLC separated fractions. However, the MALDI/TOF MS spectra
from these HPLC fractions also contained several smaller ions,
with lower masses corresponding to up to six fewer amino acids.
Prior to the Edman sequence analysis, the significance of these
ions was not clear. The first 10 amino acids from this sample gave
the sequence RQNGTVKWFN, which is an exact match for
residues 4-13 from the P. aeruginosa cold shock protein A (CspA,
see Figure 6a). The absence of residues 1-3 (MSN) is attributed
to degradation, starting during protein isolation and continuing
until the sequence was determined. However, the major component in both cells and in the initially collected HPLC fractions is
believed to be intact CspA. The expected mass, including the three
missing N-terminal residues, is 7605 daltons, whereas the major
observed mass was 7643 daltons (Table 1). The mass that is higher
(23) Dai, Y.; Liang, L.; Roser, D. C.; Long, S. R. Rapid Commun. Mass Spectrom.
1999, 13, 73-78.
(24) Holland, R. D.; Burns, G.; Persons, C. C.; Rafii, F.; Sutherland, J. B.; Lay, J.
O., Jr. Proceedings of the 45th ASMS Conference on Mass Spectrometry and
Allied Topics, Palm Springs, CA, 1997; p 1353.

Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

3229

by 38 daltons attributed to the detection of a potassium adduct

ion rather than a protonated molecule, although some other form
of protein modification cannot be ruled out.
A cold-acclimation protein was also identified as one of the
components detected in the MALDI/TOF mass spectra from
P. putida. An HPLC component was collected having a mass of
7684 daltons, also corresponding to a major ion in the spectra
obtained from cells (data not shown). The sequence observed
from this component, on the basis of the Edman degradation, was
SNRQKGTVKWFNDEK, corresponding to 14 of 15 expected
amino acids in residues 2-16 (M, the first residue is not typically
detected) from a known P. fragi cold-acclimation protein B (CapB,
Figure 6b). The observed sequence corresponds to exchange of
a lysine for a threonine in the sixth residue of the sequence. The
predicted mass for this protein, assuming no other mutations, is
7727 daltons. This is not in exact agreement with the observed
mass, 7684 daltons, suggesting other differences between this
CapB and the P. fragi CapB protein. The identification of this
protein as a CapB homologue was relatively straightforward with
both the molecular weight data and the protein sequence data.
CONCLUSION
The primary objective of this work was to isolate and identify
specific proteins associated with characteristic ions in spectra
obtained using the very rapid MALDI/TOF MS method of analysis
(9-13). This was accomplished using bacterial suspensions; ions
prominent in MALDI spectra from cells were also observed using
MALDI to analyze bacterial suspensions and subsequently in
spectra from HPLC isolated fractions. Table 1 gives a list of the

3230 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

proteins identified, their HPLC retention times, the expected

masses (based on the Edman derived sequences shown in the
figures), and the experimentally observed masses. The identity
of the proteins was deduced on the basis of the amino acid
sequences and the measured protein masses. The use of these
ions as biomarkers should be significantly enhanced by their
identification, especially since they have now been associated with
specific bacterial genes. We have identified two acid resistance
proteins, HdeA and HdeB, from both E. coli and S. flexneri and
identified the specific masses in the MALDI/TOF mass spectra
from cells (and cell extracts) associated with these two important
proteins. Similarly, a cold-shock protein in P. aeruginosa and coldacclimation protein from P. putida were identified. The same
approach could probably be applied to the detection of antibioticresistance, or heat-shock proteins, on the basis of the characterization of unique proteins detected from antibiotic- or heat-resistant
bacteria. This approach holds much promise because so many
proteins can be detected simultaneously in a single MALDI/TOF
MS experiment; only the unique or biomarker proteins need
actually be sequenced in the more time-intensive sequencing
steps. Identification of biomarker proteins will also aid the study
of bacterial physiology, as the profiles of specific proteins can be
followed under various conditions of stress.

Received for review February 8, 1999. Accepted May 5,

1999.
AC990175V