432
Electrophoresis 1997. 18, 432-442
P. Dainese ef a / .
Probing protein function using a combination of gene
knockout and proteome analysis by mass spectrometry
Paola Dainese’
Werner Staudenmann’
Manfred0 Quadroni’
Chantal Korostensky’
Gaston Gonnet’
Michael Kertesz’
Peter James’
‘Protein Chemistry Laboratory
’Computational Biology Research
Group
’Microbiology Institute, Swiss
Federal Institute of Technology,
ETH-Zentrum, Zurich, Switzerland
Recently the determination of the genome sequences of three procaryotes
(Haemophilus influenzae, Methanococcus ,jannaschii and Mycoplasma genitahum) as well as the first eucaryotic genome (Saccharomyces cerevisiae) were
completed. Between 40-60% of the genes were found to code for proteins to
which no function could be assigned. We describe an approach which con?bines proteome analysis (mapping of expressed proteins isolated by twodimensional polyacrylamide gel electrophoresis to the genome) with genetic
manipulations to study the complex pattern of protein regulation occurring in
Escherichia coli in response to sulfate starvation. We have previously described
the upregulation of eight spots on two-dimensional (2-D) gels in response to
sulfate starvation and the assignment of six of these to entries in the E. coli
genome sequence (Quadroni et al., Eur. J. Biochem. 1996, 239, 773-781). Here
we describe the identification of the remaining two proteins which are
encoded in a sulfate-controlled operon in the 21.5’ region of the E. colr
genome. Upregulated protein spots were cut from multiple 2-D gels collected
and run on a modified funnel gel to concentrate the proteins and remove the
sodium dodecyl sulfate before digestion. The peptide masses obtained from
the digests were used to search the SwissProt database or a six-frame translation of the EMBL DNA database using a peptide mass fingerprinting algorithm. A digest can be reanalyzed after deuterium exchange to obtain a
second, orthogonal data set to increase the confidence level of protein identification. The digests of the remaining unidentified proteins were used for peptide fragment generation using either post-source decay in a matrix-assisted
laser desorption ionization (MALDI) time-of-flight mass spectrometer or collision-induced dissociation (CID) coupled mass specrometry (MS/MS) with
triple stage quadrupole or ion trap mass spectrometers. The spectra were used
as peptide fragment fingerprints to search the SwissProt and EMBL databases.
1 Introduction
In the past year and a half, three complete bacterial
genome sequences (Haemophilus inj7uenzae [ 11, Mycoplasma genitalium [2], and Methanococcus jannaschii [3])
as well as a eucaryotic genome (Saccharomyces cerevisiae,
available on the internet at http:/genome-www.stanford.edu/Saccharomyces/) were completed. About 45 OIo of
the open reading frames (ORF) identified in the procaryote genome sequences of M. genitalium (470 ORFs),
H. injluenzae (1743 ORFs) and Escherichia coli (an estimated 4100 ORFs according to the Japanese genome
project http://bsw3.aistnara.ac.jp)code for proteins of
unknown function [l, 21. The main challenge arising out
of the genome projects will be to try to develop methods
to assign functions to the various ORE In this article we
describe our attempts to combine genetic approaches (by
random gene knockout or activation) with proteome
~~
~
Dr. Peter James, Protein Chemistry Laboratory,
Universitaetstrasse 16, ETH-Zentrum, CH-8092 Zurich, Switzerland,
(Tel: +41-1632-2919; Fax: +41-1632-1213; E-mail: bcmass@bc.biol.
Correspondence:
ethz.ch)
Nonstandard abbreviations: AMU, atomic mass units; CID, collisioninduced dissociation; MS/MS, fragmentation analysis using coupled
mass scanning devices; ORF, open reading frame; PSD, post source
decay analysis; SSI, sulfate starvation-induced; TOF, lime of flight;
WWW, World Widc Wcb
Keywords: Proteome / Gene knockout I Fingerprinting / Sulfate starvation / Mass spectrometry
0 VCH Verlag\gcsellschaft mbH,
69451 Weinhelm, 1997
analysis (mapping changes in gene expression by 2-D gel
electrophoresis and mass spectrometry) in order to
assign functions to proteins involved in the sulfate starvation response in E. coli.
Bacteria must be able to respond rapidly to changes in
their environment since they are relatively immobile.
They can modulate the expression of individual genes o r
large groups of genes, regulons, which are sets of operons with a common regulator, in response to the needs
of the organism. The entire set of genes responding LO
an environmental stimulus is termed a “stimulon” [4].
Such global regulation systems are well established in
E. coli for the assimilation of carbon, nitrogen and phosphorus [5, 61 and recently for sulfur [7]. Sulfur comprises
ca. 1 % of the dry weight of a cell [8] and is assimilated
primarily from sulfate in the cysteine biosynthetic
pathway. Sulfate uptake and assimilation is under the
control of the “cys regulon” [9]. Sulfate is bound by a
periplasmic sulfate binding protein and moved across
the cytoplasmic membrane by two channel-forming
membrane proteins which are bound to a cytoplasmic
nucleotide binding subunit. The sulfate is reduced to sulfite and then to sulfide before reacting with O-acetylserine to form cysteine. If the sulfide concentration in a
cell drops, the amount of 0-acetylserine rises, as well as
N-acetylserine, which is formed by an irreversible N - 0
migration of the acetyl group. Full expression of the cys
regulon requires the presence of N-acetylserine and the
transcriptional activator protein CysB which positively
regulates the genes of the cys regulon [lo]. In soil how0173-0835/97/0304-0432
$17.50+.561/0
Elrcrrouhoresis 1991. 18. 432-442
ever, a high percentage of sulfur is present in an organic
form such as sulfate esters, sulfamates, amino acids and
sulfonic acids [Ill. In order to study how E. coli survives
in soil under sulfate starvation conditions, we grew the
bacteria in vitro with ethanesulfonate as the sole sulfur
source. We have previously shown that eight proteins are
induced by a factor of a least 2 and have identified six of
them [12]. We describe here the identification of the
other two proteins and outline the evidence that a sulfate starvation regulon exists in E. coli.
2 Materials and methods
2.1 Materials
Acrylamide, N,N-methylenebisacrylamide and carrier ampholytes for 2-D electrophoresis were purchased from
BDH (Poole, England); CHAPS and NP-40 were from
Sigma (Buchs, Switzerland); Coomassie Brilliant Blue
(Serva Blue G ) was from Serva (Heidelberg, Germany);
SyPro Orange and Red were from Molecular Probes
(Eugene, OR, USA); ImmobilineTM strips were from
Pharmacia (Uppsala, Sweden). All other reagents for 2-D
electrophoresis were the highest purity grade available
from Fluka (Buchs, Switzerland). Fluorotrans PVDF
membrane was obtained from PALL (Muttenz, Switzerland) and 0-octylglucopyranoside was from Pierce (Rockford, IL, USA). Sequencing grade modified trypsin was
purchased from Promega (Zurich, Switzerland) and
DNase and RNase were from Boehringer (Mannheim,
Germany). All HPLC solvents used were from RiedeldeHaen (Seelze, Germany).
2.2 Bacterial culture and cell extraction
Escherichia coli MC4100 (F- aruD139 A(argF-lac) U169
rpsL1.50 relAl deoCl p t s R 5 rpsRflbB5301) [13] was obtained from the laboratory collection. Bacteria were cultivated in a sulfur-free, synthetic glucose-salts medium as
previously described [7], with the addition of either inorganic sulfate (500 pm) or ethanesulfonate (500 pm) as
the sole sulfur source. The culture were grown aerobically on a rotary shaker (180 rpm) at 37"C, and growth
was monitored spectrophotometrically at 650 nm. Cells
were harvested in the mid-exponential phase (A,,,=0.5)
by centrifugation (7000 X g , 10 min, 4°C) and washed
with 50 mM Tris/HCl, pH 7.0. They were then resuspended in the same buffer (0.8 g wet mass per mL) and
ruptured by three passages through a chilled French
pressure cell at 135 MPa. Ten I.ILof 10 mM Tris/HCl, pH
7.5, was added per 200 pL of pellet, followed by 20 pL of
1% w/v SDS, 150 mM DTT. The solution was kept at
95°C for 5 min and then DNase I (50 pg/mL) and RNase
A (10 pg/mL) were added and incubated for 30 min at
37". Cell debris was removed by centrifugation (12 000
X g, 30 min, 4°C).
2.3 2-D gel electrophoresis
The first-dimensional Immobiline strips were run in batches of 20 on a Multiphor I1 system. The Immobiline
strips were reswollen overnight in 8 M urea, 2% CHAPS,
10 mM DTT, 0.8% carrier ampholytes pH 4-8. Typically,
400 p of proteins were loaded on each Immobiline strip
Probing protein [unction by gene knockout and proteome analysis
433
when using the Pharmacia cup system. The strips were
focused at 300 V for 3 h to allow the samples to enter
the gel, then the voltage was ramped up to 3500 V over
6 h and run at 3500 V for 24 h. Large loadings were carried out using a gel rehydration cassette as described by
Rabilloud [14]. After the focusing was complete, the
strips were equilibrated for 20 min in 50 mM Tris/HCl,
pH 6.8, 6 M urea, 25% glycerol, 0.2% SDS, 30 mM DTT,
before changing the buffer and incubating for 5 min in
50 mM Tris/HCI, pH 6.8, 6 M urea, 25% glycerol, 0.2%
SDS, 65 mM iodoacetamide. The strips were transferred
onto 12% polyacrylamide gels (Laemmli system). The
second-dimensional gels were run in a 40 L tank at
12-15°C overnight at a constant current of 400 mA
(using a running buffer of 25 mM Tris, 200 mM glycine,
0.19'0 w/v SDS) using an Iso-Dalt apparatus (Hoefer, San
Francisco, CA, USA). Six batches of gels were run, each
from a different preparation of E. coli to ensure the
reproducibility of the sulfate starvation induction effect.
Gels were stained with Coomassie Brilliant Blue
according to the protocol of Schagger and von Jagow
[15], since it produces a very clear background, suitable
for scanning densitometry. Gels were fixed overnight in
methanoUacetic acid/water (50/10/40 v/v), stained in
0.025% w/v Serva Blue G in 10% v/v acetic acid for 3 h
and then destained in 10% v/v acetic acid. Silver staining
was carried out according to Doucet and Trifaro [ 161.
The gels were fixed in 40% ethanol, 10% acetic acid overnight and then washed with Millipore UltraP water (3 X
20 min) before sensitization for 30 rnin with a solution
of 5 mg/L DTT (30 rnin). Silver impregnation was carried out by soaking in 0.1% AgNO, for 30 rnin before
washing twice with water for 30 s. The gels were developed with a solution of Na,CO, (30 g/L) with 300 pL/L
(37% v/v in water) formaldehyde. Gels were then placed
in a stop solution of 1% acetic acid for storage. Wet gels
were scanned in a Personal Densitometer (Molecular
Dynamics, Sunnyvale, CA, USA) and image analysis and
spot matching were performed using the Investigator
software package (Millipore, Bedford, MA, USA) on a
Sun workstation. The p l and M, abscissa were calibrated
using the known values from two proteins, alkyl hydroperoxide reductase C-22 and the sulfate-binding protein
as described in [12].
2.4 Protein elution, concentration and electrotransfer
onto PVDF membrane
Individual proteins were excised from multiple 2-D gels
of E. coli grown under sulfate starvation conditions and
concentrated to single sharp bands using a funnelshaped gel electrophoresis device (Dainese, P. et al.,
unpublished). Proteins were then electroblotted onto
PVDF membranes in a semidry apparatus (Hoefer) in a
buffer containing 50 mM Tris/HCl, 192 mM glycine,
0.02% w/v SDS, 10% v/v methanol, 2 mM DTT, for 1 h
at approximately 1.2 mA/cm2. Proteins were visualized
on the membranes by a 5 min incubation in 0.1% w/v
Serva Blue R in 50% v/v methanol, followed by destaining in 70% v/v methanol for 5-10 min.
2.5 Protein digestion
Dried PVDF membrane slices were cut into small pieces
(1 mm2) and equilibrated for 1 h at room temperature in
434
P. Dainese
Electrophoresis 1997, 18, 432-442
el a / .
10 pL of 1O/o w/v 0-octylglucopyranoside, 100 mM ammonium bicarbonate (pH 7.8). Digestion was initiated by
the addition of 1 pg of Promega trypsin (1 pg/pL in the
same buffer) and carried out for 15 h at room temperature. Digestion was stopped by adding 1 ILLof 2% v/v
TFA to the sample. The supernatant was collected and
the membrane pieces were washed twice with 10 yL
0.1% v/v TFA and the washes were pooled with the
supernatant .
2.6 MALDI-MS and post-source decay (PSD) analysis
MALDI time of flight (TOF) mass spectra were accumulated using a Voyager Elite (Perseptive Biosystems, Framingham, MA, USA). Samples were acidified with 10%
TFA to lower the pH to below 3 for cocrystallization
acid (5 mg/mL in 50%
with a-cyano-4-hydroxy-cinnamic
acetonitrile, 50% 0.1% TFA in water) on a 100 position
sample tray. The crystals were washed (three times) by
covering the spot with a drop of ice-cold water (for 5 s),
which was then removed by suction using a fine pipette.
Samples were analyzed in pulsed extraction reflector
mode using an accelerating voltage of 20 kV, a pulse
delay time of 75 ns, a grid voltage of 55% and guide wire
voltage of 0.05%.
After the 100 positions had been analyzed, each sample
was wetted with 1 pL of deuterium oxide and allowed to
dry. This was repeated four times in an airtight glove box
in which the atmosphere was saturated with deuterium
oxide and kept under a slight overpressure of nitrogen.
The glove box was located immediately above the
sample inlet to the mass spectrometer from where the
target could be moved into the instrument without exposure to the air. PSD spectra were accumulated from the
same spots using a 50% higher incident laser power and
setting the timed ion selector to the mass of interest and
varying the mirror ratio from 1 to 0.02. The guide wire
voltage was lowered to 0.02% for mirror ratios below 0.3
and spectra were accumulated for 64 to 256 shots per
mirror ratio setting.
2.7 Automated peptide fragmentation by collision-induced
dissociation (CID)
Digestion mixtures were separated by reversFd-phase
HPLC on a capillary column (C,,, 5 yL, 300 A, 280 X
0.05 mm) from LC Packings International (Zurich, Switzerland) directly connected to a Finnigan MAT (San
Jose, CA, USA) TSQ 700 triple quadrupole mass spectrometer equipped with an electrospray ionization
source using a coaxial flow of 1.5 pL/min methoxyethanol as a sheath liquid. The total flow was 3 yL/min
and the column was washed extensively with solvent A
(0.1 Yo v/v TFA in H,O) before running a 60 min linear
gradient from 0 to 70% solvent B (80% v/v acetonitrile,
0.08% v/v TFA). A program, Autofrag, which automates
the collection of CID fragmentation spectra from
unknown samples, was written in Finnigan MAT instrument control language [17]. The program was subsequently modified to incorporate features of a similar program presented by Dr. Terry Lee at the 1993 American
Society of Mass Spectrometrists meeting [18]. The program monitors the masses of the peptides eluting from
the column, using the first quadrupole Q1 to scan the
mass range from 500 to 2000 atomic mass units (amu)
every 4 s (with 2 mTorr argon in 42 but with the collision offset voltage set to zero). The program switches to
MS/MS mode every time a signal lasting more than two
scans is detected with a signal/noise ratio > 5. Quadrupole Q1 filters out the selected ion which undergoes fragmentation in the second quadrupole, filled with a collision gas (argon) to a pressure of 2 mTorr. The program
returns to scanning Q1 after 5 MS/MS scans so that less
intense, coeluting peptides can also be analyzed. The collision offset voltage (the voltage for accelerating the ions
in the collision chamber, Q2) is automatically adjusted to
a value determined by the mass of the ion selected. The
resulting fragments are analyzed with the third quadrupole 4 3 [19], scanning the mass range from 50 to 2000
amu in 3.0 s . This procedure allows both parent ion
mass measurement (for protein mass fingerprinting) and
sequence analysis by fragmentation (for peptide mass fingerprinting) to be carried out during the same HPLC
run. The program has an optional lookup table which
contains masses of common contaminants such a8
trypsin and keratin fragments and nonpeptidic ions
arising from the gel. These masses are not used for carrying out MS/MS analysis.
3 Results
In order to search for proteins expressed at low levels
which are induced by sulfate starvation, we changed
from using carrier ampholyte-based tube gels in the firsl.
dimension to a flat-bed Immobiline system. Using the
gel rehydration method of sample loading described by
Rabilloud [14], it was possible to increase the loading
from 100 pg to 10 mg per strip. The 2-D images obtained
using a first dimension of carrier ampholytes in tube gels
were very different from the ones obtained using Immobiline strips in the first dimension; therefore, we
repeated the sulfate starvation study with the new
system. Figure 1 shows a 2-D gel of wild-type E. coli
grown in the presence of 500 pm inorganic sulfate (left
panel) and wild-type E. coli grown with 500 pm ethanesulfonate (right panel). Eight spots were found which are
upregulated by a factor of at least 2, corresponding to
the ones previously observed [ 121. Interestingly, several
of the spots had changed their relative positions and
spots 2 and 3 showed a reversal of apparent p l values.
The spots were excised from sixteen gels and concentrated using the funnel gel system and then digested.
The amount of each protein used for identification (estimated from the integrated intensity of Coomassie
staining of the proteins in comparison with that of a
dilution series of bovine serum albumin) was between
1-50 pmol. The resulting peptides were extracted into a
final volume of ca. 20 pL. The digests were analyzed
according to a three-tier mass spectrometry approach to
data collection for protein identification and characterization (represented schematically in Fig. 2).
3.1 First-level mass spectrometric analysis: peptide mass
fingerprinting
One pL of the protein digest was used for analysis by
MALDI-TOF-MS. Reproducible good spectra were ob-
Electrophoresis 1997, 18. 432-442
Probing protein function by gene knockout and proteome analysis
435
Figure 1. 2-D PAGE mapping of Escherrchia coli grown in the presence or absence of inorganic sulfate. E. cull was grown with 500 pm sulfate
(left panel) or 500 pm ethane sulfonate (right panel) as the sole sulfur source. All the proteins which were induced during growth with ethane
sulfonate by more than a factor of 2 X are labeled. Two proteins which were expressed at the same level under both growth conditions are
labeled as EC9A and EC19A.
tained at the 100 femtomole level for protein digests
containing octyglucoside. The sample was pipetted,
together with an equal volume of matrix, onto the surface of a 100 position target and allowed to dry. The
spectra were accumulated in an automated overnight
run and the digests giving poor or no spectra were reanalyzed manually the next day. The stability of the mass
calibration (+ 0.3 amu) was high enough that no
internal calibration was required. The spectral accumulation used less than 5% of the material deposited on the
target (which could be kept for weeks and reexamined at
will). The samples were then remeasured after deuterium exchange and the two sets of peptide molecular
masses obtained (the native and deuterated peptide
mass fingerprints) were used to search protein (SwissProt) and nucleic acid sequence (EMBL) databases
using the program MassSearch [20, 211. Figure 3 shows
the MALDI-TOF mass spectrum of the trypsin digest of
spot EC9a, which was used as a control, since the level
of its expression was the same in the presence or
absence of inorganic sulfate. Table 1 shows the MassSearch output for the single digest. The confidence level
of the score, the difference between the correct match
and the next nonrelated protein was 15.9. In order to
confirm the identification as elongation factor Tu, a
second data set, usually obtained by deuteration of the
first, was used as an orthogonal fingerprint. Table 2
shows the increase in confidence (from 15.9 to 140.3)
achieved by using two data sets (here with two digestions). If the protein was positively identified, the relevant database entry was downloaded and examined in
detail. All the peptide masses observed were checked
against the sequence, allowing for partial digestions and
modifications such as oxidation, deamidation, carbamylation, etc. The masses which did not match were used to
search the database again to check if two (or more) proteins were present in the same spot.
The first stage of analysis, peptide mass fingerprinting,
conclusively identified three of the proteins as sulfate
binding protein (spot 2), cysteine synthase A (spot 5 )
and alkylhydroperoxide reductase (spot 8) in the SwissProt database, whilst the fourth protein, the j7iY gene
product, a cystine binding protein (spot 7), could only be
identified as an entry in the DNA database using a dual
data set. As a general rule, if a protein was present in a
protein sequence database, a set of six masses from a
single digest was sufficient to identify the protein correctly with the levels of mass accuracy achieved using
automated data collection. For proteins in the six-frame
translation of the DNA database, a dual digestion with
four masses per digest was found to be the minimum
required under the same data accumulation conditions.
Dual data sets do not require more material, since a deuterated data set can be obtained by reusing the samples
on the MALDI target. After single, dual and high mass
accuracy searches, four proteins remained unidentified.
These were used for the second-level analysis.
3.2 Second-level mass spectrometric analysis: peptide
fragment fingerprinting
After peptide mass fingerprinting, 19/20 of the protein
digest still remained and 1/20 was on the MALDI-TOF
436
P. Dainese et
Electrophoresis 1997. 18, 432-442
a/.
digests showed peptides that were sufficiently mass-separated that one could attempt sequencing by daughter
ion analysis in a reflectron TOF-MS using MALDI. A
second aliquot of 1 pL was used for PSD [22]. The
parent ion was selected by a timed ion gate which can
resolve ions separated by a least 40 amu from the next
ion (up to a mass of ca. 2000). PSD analysis is useful for
poor digestions when a few peptides, well separated by
mass, are obtained, though at least 100-200 femtomoles
of material is required. The high energy fragmentation
patterns differ from those obtained using a low energy
regime such as in an ion trap or triple quadrupole MS.
The spectra could be used directly with the high energy
version of the Sequest program [23] for database
searching, or interpreted manually, and a peptide tag
used as input to the Peptidesearch program [24]. Figure 4
shows the PSD spectrum of peptide mass 1275.6 from
the control protein, EC19a. Database searches using the
uninterpreted spectrum with high energy Sequest and
using a partial TAG sequence with the Peptidesearch
program identified the protein as E. coli B-lactamase.
target after fingerprint analysis (since only a few percent
of the material loaded was actually consumed during
data accumulation). On average, about one in four
+
~
FINGERPRINT
-l Q
POSlTlV
I
1
1
I
DEUTERATE
DIGEST
I
Since none of the remaining four protein digests gave a
peptide mass distribution suitable for PSD analysis, the
digests were used for auto-HPLC-MUMS peptide fragment fingerprinting. Half of the remaining 18/20 was
used for peptide fragment fingerprinting by HPLC-MSI
MS. The samples were loaded into 50 pm internal
diameter capillaries packed with C-18, 300 A, 5 pvn
reverse-phase material and eluted by a gradient of acetonitrile in water into the triple stage quadrupole or ion
trap mass spectrometers for automated on-line HPLC
MSIMS data collection. This procedure generated (on
average) partial to complete sequence spectra for >70%
of the peptides in the digest extract. Figure 5 shows the
auto MS-MS run for protein 3. The masses of the
eluting peptides are listed above the respective total ion
current (TIC) peaks. The spectra for each of the ca. 40
masses chosen for MSIMS were averaged and written to
separate files for subsequent database searching with the
Sequest program [25]. Table 3 shows the result of a
single peptide fragment fingerprint search from protein
spot 3. Four other MS/MS spectra matched with this
A
SEARCH
SEARCH
MANUALLY
SEARCH
Figure 2. Logical flow diagram of the strategy used for protein identification.
1962.1
20,000_)
I
1
1233.4
21 17.5
1781.2
2240.1
2729.5
I 2240.1
I
I
1,000
1,500
I
2,000
Mass (m/z)
Figure 3. MALDI-TOF spectrum of the tryptic digestion of protein EC9a.
I
I
2,500
I
3,000
Probing protein function by gene knockout and proteome analysis
Elecrrophoresis 1997, 18, 432-442
Table 1. Protein identification using single digest data")
Number
Scoreb'
n
k
AC
DE/OS
1
79.5
14
3
2
79.5
14
3
3
63.6
6
2
4
63.1
7
2
5
62.4
5
2
PO2990
Unmatched
P21694
Unmatched
PO973 1
Unmatched
P32800
Unmatched
401360
Unmatched
Elongation factor Tu, E. coli
weights: 1233.4; 2240.8
Elongation factor Tu, S. typhimurium
weights: 1233.4; 2240.8
Protein HXLF5.Cytomegalovirus
weights: 1962.1; 2117.5: 2240.8
CRTl protein. S. cerevisiae
weights: 1233.4; 1781.2; 1962.1
Aliphatic amidase. R . eythropolis
weights: 1781.2; 2117.5; 2240.8
a) The table shows the Masssearch output using the tryptic peptide masses of protein EC9a measured by MALDITOF, shown in Fig. 3. Searching SwissProt release 32 using average masses. Scores lower than 60 are probably
not significant. The average tryptic fragment masses used were: 1233.4. 1781.2, 1962.1, 2117.5, 2240.8
b) Score is the inverse logarithm of the probability of the matches occurring at random; n is the number of peptides in the matching protein which have masses between the lowest and highest match used in the search; k is
the number of peptides showing matches. AC is the accession number of the matching protein in the SwissProt
database. DE is the SwissProl annotated description of the protein.
Table 2. Protein identification using orthogonal data sets: Masssearch output for EC9a"'
Number
Score
n
k
n
k
AC
DElOS
1
236.9
14
3
5
5
PO2990
2
236.9
14
3
5
5
P21694
3
162.0
12
3
6
4
P43926
4
104.1
12
4
7
3
P29542
7
96.6
7
2
4
3
P35647
Elongation factor Tu, E. coli
Unmatched tryptic: 1233.4; 2240.8
All AspN weights matched
Elongation factor Tu, S. typhimurium
Unmatched tryptic: 1233.4; 2240.8
All AspN weights matched
Elongation factor Tu, H. influenzae
Unmatched tryptic: 1233.4; 2240.8
Unmatched AspN: 1289.4.
Elongation factor Tu, S. ramocissimus
Unmatched tryptic: 2240.8
Unmatched AspN: 1196.3; 1289.4
Hemagglutinin 1, E. corrodens
Unmatched tryptic: 1962.1, 2117.5, 2240.8
Unmatched AspN: 598.6; 1196.3
a) Searching SwissProt release 32 using average masses. Scores lower than 90 are probably not significant. The
tryptic fragment masses used were: 1233.4, 1781.2, 1962.1, 2117.5, 2240.8, and for AspN the fragment masses
were: 598.6, 929.1, 1071.3, 1196.3, 1289.4. Abbreviations as in Table 1.
ARG
N
0
r
r
300
0
r
X
v)
20-
CI
S
3
s
10-
I
200
400
600
800
1000
1200
Mass m/z
Figure 4. PSD spectrum of peptide mass 1275.6 from the control protein, EC19a. The sequence tag used for
searching is indicated. The ion series can be easily identified since all occur in groups showing a characteristic mass
separation (a+28=b, b+17=c). The protein was identified by Peptidesearch and high energy Sequest as p-lactamase.
437
43 8
P. Dainese
1001
PI
al.
Electrophoresis
724.5
ol
MS Scan
80-
‘x50
10+200
I
8
530.2
60. 573.2
Select only 725
-64
1997, 18. 402-442
I
1724
MS/MS Scan
I
486
436
I
4
1
I I
40-
20-
I,
600
1000
I
1400
100 200 300 400 500 600 700 8
1800
masskharge
ION CURRENT TRACE
Figure 5. On-line HPLC auto-MS and MS/MS peptide fragment fingerprint accumulation for the tryptic digest of SSI protein #3. The bottom
panel shows the intensity of ions entering the mass spectrometer from the HPLC against time, expressed as scan numbers (scanning from mass
500 to 2000 in 4 s). The masses of the eluting peptides are given above the peaks. In this example, a peptide of mass 724.5 in scan 56 (top left)
was identified by the Autofrag program as a candidate for sequence analysis and was then isolated and collisionally activated to give an MS/M!I
or fragmentation spectrum (top right). Manual interpretation of the spectrum gave the sequence Thr-His-Pro-Val-Ser-Gly-Lys.
Table 3. Sulfate starvation-induced protein #3 is found in the databases using peptide fragment fingerprintinga’
Sequest search result
Number
(M+H)+
1
2
3
ORFl
4
5
deltCn
Ions
Access #
775
715
715
0.0000
8/11
0.1561
0.2272
15/25
91 18
tauD
4-Coumarate-CoA ligase 1
Transposon TXI, hypothetical protein
715
715
0.2655
0.3899
8/19
1113 1
G2-specific protein kinase
Phosphoribosylamine-glycine ligase
a) Sequest output using MS/MS spectrum of tryptic mass 775 from sulfate starvation-induced protein
#3 to search the database.
region of DNA in the 8.5‘ region of the E. coli chromosome. Eight MS/MS spectra from spot 1 matched an adjoining region around 8.5‘. Figure 6 shows these peptides
aligned against a translation of the genomic sequence.
Peptides from the auto HPLC-MS/MS runs of spots 4
and 6 were subsequently found grouped together in the
21.3’ region of the E. coli chromosome (see Table 4).
3.3 Third-level mass spectrometric analysis: subtractive
MS/MS
If the protein was successfully identified, the 9/20 of the
digest remaining after peptide mass and peptide frag-
ment fingerprint analysis was analyzed by HPLC-MS/
MS using a modified version of AutoFrag called ModFrag. The program ignores all the masses in a userdefined list (e.g. the tryptic masses predicted from the
genome sequence) and sequences only the unknowns.
The resulting MS/MS spectra coming from ions that are
not predicted from the known sequence are used to
research the databases in order to detect peptides
coming from comigrating proteins, or to detect possible
DNA sequencing errors leading to amino acid substitutions, and reading frame shifts. The nonmatching spectra
remaining after both searches are then manually interpreted to give a complete sequence [19] or used for a.
Probing protein function by gene knockout and proteome analysis
Elerrruphurrsrs 1991. 18. 432-442
1
2
3
V
V
1
2
3
P
W R I K P Q P N R
R G V S N L S R T G
R
E
A K E S G E P W T G V
L K K A E N R G L A
' R K R R T V D W R
1
2
3
K
S
S
A
W
G
L
Q
L
S
T
D N T F
P T T P L R Q H L C -
A
R
G
A
E
Q
H
R
A
G A G F R R R A N R Q P R F Q P V S G C
A L A S G D V Q I G N L G S S P L A V A
R W L Q A T C K
' R L Q
1
2
3
1
2
3
N T L L A A L A F I A F Q A Q A
f
1
T H F L P H W H S S L F R H R R ' T S ' H T S C R T G I H R F S G T G G E R H
R
L
a
-
-
S Q P T G A D * S L L A G V K T G * L R
.A .S SQ :Q *V ;P ZI BE l
V F
G N S E
P A N R C R L K S S C W R Q N W V T P K -
3
S A G G K E N Y Q Q T G R S D W Q T H R
A L V V K K T I S K P E D L I G K R m
R W W * R K L S A N R K I ' L A N A S P -
1
R
1
2
T
2
3
1
Y
H
R
'
I
2
L
3
V
*
L
S
V
S
T T H Y S L A G G T E T L G
P P P T T A W L A A L K H W G
V H H P L Q P G W R H ' N T G A
A S G D C R T C S R P R L S L P
K P G Q V E I V E P A A A R D Y R C L N P G K W R L ' N L Q P P A I I A A W T
R
1
2
3
G S G E I L M
A A G R Y * W
Q R G D I
1
K
T
R
2
1
2
3
T
1
L
2
3
1
2
3
R
A
R
D
3
-
G
V
C
L
L
M
C
S
L
G H R R L T P W K
G T G G ' R P G K -
R C + P I L N R S G S G A R Q R W
Q G V D R P * T G R A V G R A N A G K V L T D S E Q V G Q W G A P T L D -
S G W C A K I L P R N I L R S ' K R S
L G G A Q R F C R E T S ' G R E S V R
A F A
W V V R
V
K A P S M L S N R T L L T Q T C G ' N
' K R H R C S A T V H C ' P R R V A E T K S A I D A Q Q P Y I A N P D V W L K Q -
S R K T
A G K H
S
Q
A N W R V * A A C L K V T F P
Q T G A F K R R A ' R ' R S R L A R L S G V P E G D V P G
Figure 6. Alignment of the fragmentation spectra matches found using
Sequest using the tryptic digest of SSI protein #I with the three forward reading frames of a preliminary DNA sequence from the E. coli
8.5'-region. The fragmentation spectra from the automated HPLCM U M S of the tryptic digest of SSI protein # I were matched against
our E. coli database using the program Sequest. The peptides produced fragmentation spectra which matched amino acid sequences
(indicated by shaded boxes) in all three forward reading frames of an
unpublished stretch of DNA sequence from E. coli.
peptide tag seaarch using the method of Mann [24]. This
helps to pick up modified peptide masses such as those
due to post-translational modifications. None of the
eight proteins analyzed showed any unexpected masses.
3.4 Loss of specific function by random gene knockout
In an attempt to further define the functions of these
last four proteins, an independent approach was taken.
Random mutants were generated using a phage carrying
a promoterless B-galactosidase gene which randomly
inserts into the E. coli chromosome. The resultant fusion
strains were screened for mutants showing increased
B-galactosidase activity under sulfate starvation conditions. If the phage inserts into a gene the corresponding
protein spot on a 2-D gel will disappear. Figure 7 shows
a 2-D gel of wild-type sulfate-starved E. coli (left panel)
and of the mutant strain, 108 (right panel). The insertion
site of the phage was found to be in the 8.5' region of
the chromosome in the middle of the ORF corresponding to that identified by peptide fragment fingerprinting of spot 3. The insertion mutants which showed
genes under sulfate control were then screened using a
series of sulfur sources in the media to find which substrates they could grow on. In seven of the nine mutants
analyzed, the phage had integrated in the 8.5'-region,
three of them into the region coding for the spot 3 protein. Interestingly, mutant 108 could grow using ethanesulfonate but not taurine as the sulfur source.
4 Discussion
All bacteria require sulfur and phosphorus for growth,
and these are usually supplied in laboratory growth
media in the form of inorganic phosphate and sulfate.
The growth of E. coli under phosphate-limited conditions
leads to an increased expression of the pho regulon, an
ensemble of 81 or more proteins [ 6 ] ,many of which are
known to be directly involved in phosphate metabolism
[27]. A similar negatively-regulated system was proposed
to exist for sulfur metabolism, since a set of proteins
(SSI proteins) can be seen by 2-D electrophoresis to be
upregulated when E. coli i s grown using compounds
other than cysteine or sulfate as the sole source of sulfur
[7]. We have recently described the identification of six
SSI proteins seen to be upregulated on Coomassie bluestained 2-D gels [12]. A similar sulfate response is shown
by Pseudomonas putida and Staphylococcus aureus, which
exhibit 14 and 10 SSI proteins, respectively [7]. The similarities suggest that mechanisms for the compensation
of sulfate starvation might be widespread among bacteria. By analogy with the phosphate starvation response
system, we believe that these proteins are involved in
the assimilation of organic sulfur sources when inorganic sulfate is scarce.
Eight proteins were seen to be upregulated when comparing Coomassie-stained 2-D gels of E. coli grown in
the presence of ethane sulfonate instead of sulfate as a
sulfur source. We have described the identification of six
Table 4. Peptide fragment fingerprints from sulfate starvation-induced proteins 4 and 6 determined by
MS/MS which match in the E. colt genome 21.6' regiona'
Protein spot #
number
Peptide mass (MH+),,
Matching sequence in genome
SSI 4
716.9
119.9
1069.5
1254.6
133.9
890.1
909.1
FDSPAXK
AAYSGAXK
TXXDXXPER
DVQVPDXXS LR
AQAAFAR
TDSVGQQR
ETVDFNGK
SSI 6
a) X indicates Ile or Leu since they are isobaric.
439
440
P. Dninese
el
ol.
Electrophoresis 1997, 18, 432-442
Figure 7. 2-D PAGE mapping of Escherichia coli wild type and 108 mutant grown in the absence of inorganic sulfate. E. coli wild type (left panel)
and the 108 mutant (right panel) were grown with 500 um ethane sulfonate as thc sole sulfur source. The magnified insets show the presence of
SSI protein 3 in the wild type and its absence in the mutant.
of these from 2-D gels using tube gels with carrier
ampholytes in the first dimension. In order to identify
the remaining two proteins we switched to using Immobiline strips as the first dimension to increase the
amount of material which could be loaded. Since the
2-D patterns obtained were different we had to repeat
the identification of all the SSI spots as well as the triangulation markers used as markers for gel comparisons.
Four of the SSI spots could be identified by peptide
mass fingerprinting using MALDI spectra: SSI spot 8
was found to be alkylhydroperoxide reductase; SSI spot
2, sulfate binding protein; SSI spot 5, cysteine synthase
A; and SSI spot 7, cystine binding protein. This was in
agreement with our previous results obtained by HPLCMS electrospray on a TSQ MS. The MALDI data accumulation, however, took only 15 minutes whereas the
HPLC-MS required 7 h. The four remaining spots, SSI 1,
3, 4, and 6 could not be identified, even using dual data
set searching.
The final four spots were analyzed by HPLC-auto-MS/
MS to obtain peptide fragment fingerprints. Matches for
peptide MS/MS spectra were found for all four proteins
in this way. However, if peptide fragment fingerprinting
could find these proteins, why had the peptide mass fingerprinting failed? The answer was immediately obvious
when one considered Fig. 6. The reading frame was
shifting so often that a high score could never be obtained in a single reading frame stretch. Spots 1 and 3
were found in the 8.5’ region (between 384.600-385.562
and 386.339-387.166 kbp, respectively) of the E. coli
chromosome and spots 4 and 6 in the 21.5‘ region
(between 993.944-994.517 and 991.500-992.643 kbp,
respectively). The E. coli DNA sequence used for the
searches was downloaded from the Japanese E. coli
genome project group’s WWW site at htp://bsw3.aistnara.ac.jp. All four of these SSI protein-encoding ORFs
had not been found (they were not in the ORF annotation list maintained at the site). The eight proteins seen
to be upregulated by comparing Coomassie-stained 2-13
gels can be divided into four functional groups: (i) SSI
spot 8, alkylhydroperoxide reductase, is a general stressrelated protein. (ii) SSI spot 2, sulfate binding protein,
spot 5 cysteine synthase A and spot 7, cystine binding
protein, form part of the cys regulon under the control
of CysB. (iii) SSI spots 1 and 3 are proteins encoded by
a region close to the hemB (porphobilinogen synthase)
gene. This region has been shown to contain four open
reading frames, tauA, B, C and D [27], where TauA and
TauD are SSI spots 1 and 3 respectively. (iv) SSI spots 4
and 6 are proteins encoded by a region between the
pepN (aminopeptidase N) and pyrD (dihydroorotate oxidase) genes and are separated by the ycbE gene, an O R F
encoding a hypothetical ABC transporter protein.
In order to further define the functions of the SSI proteins, random mutants were generated using the
hplacMu9 system. Nine mutants were isolated whiclh
showed sulfur-regulated B-galactosidase activity [27]. 2-D
gel analysis of mutant strain MS-108 showed that SSI
Electrophorrsis 1997, 18, 432-442
spot 3 disappears, indicating that the phage had integrated into the tauD gene. This was confirmed by DNA
sequencing. The insertion sites of the phage in the other
mutants showed all occurred in the 8.5’ region (mutant
61 was in the tauB gene; 11, 15, and 43 in the tauC gene
and 74, 82, 104, 108 and 115 in tauD). Inspection of the
sequence of the tauABCD region showed that there was
a single promoter before tauA and that the region was
probably transcribed as an operon. TauABCD showed
many features characteristic of ABC-type transporter systems such as a periplasmic substrate binding protein, a
channel-forming membrane protein and a cytoplasmic
nucleotide binding protein [28]. A comparison of the
tauA-encoded sequence with the N-terminal of TauA
(SSI spot 1) showed that a signal peptide had been
cleaved off, indicating a periplasmic location. This was
consistent with TauA being a periplasmic binding protein. TauB was found to be homologous to known ATPbinding proteins of ABC transporter and TauC showed a
hydropathy plot characteristic of a membrane protein.
TauD, SSI spot 3, shows homology to tdfA dioxygenase
of Alcaligenes eutrophus which catalyzes the conversion
of 2,4-dichlorophenoxyacetate to 2,4-dichlorophenol
and concommitantly, glyoxylate and a-ketoglutarate to
succinate and carbon dioxide. Studies on taurine degradation in other microorganism show it is usually oxidatively deaminated to sulfoacetaldehyde and then cleaved
to sulfate and acetate. Thus TauD may be responsible for
oxidatively desulfonating taurine to release sulfite which
enters the cysteine biosynthesis pathway. Disruption of
tauD and the other tau genes only affected the ability of
the bacterium to grow, using taurine as a sulfur source,
and did not affect the utilization of alkylsulfonates; tau
is therefore thought to represent a sulfur-regulated
operon involved in the metabolism of taurine.
The genes encoding SSI spots 4 and 6 were found in the
same region of the chromosome, on either side of an
ORF coding for a hypothetical ABC transporter. This
region also showed great similarity to an ABC transporter operon. This second sulfur-controlled operon containing SSI 4 and 6 may be responsible for the uptake of
alkylsulfonates since disruptions in the tau genes led to
an inability to grow with taurine as the sulfur source but
did not affect the uptake of ethanesulfonate. Gene inactivation analysis should be able to provide the data to
help answer this question.
The results that we present here show that proteome
analysis of gene activation in response to environmental
factors, in combination with gene knockout, is a
powerful technique to obtain information about the
function of unknown ORFs in genome sequences. The
screening for the loss of a specific function by random
gene knockout ensure that changes visible by 2-D PAGE
are not just due to random upregulation of proteins or
to a generic stress response. Proteome analysis will not
only help to find functions for the ca. 40% of the
genome ORFs which have no homology to known genes
but will also allow a broader understanding of genome
organization, especially the coordination of global
responses. We are extending our proteome analysis
studies to include gene knockout or permanent activation of specific genes in order to find which proteins are
Probing protein function by g e n e knockout and proteome analysis
441
coregulated as part of a global response. The sensitivity
of protein identification is increasing all the time [29]
and the use of higher loading techniques [14] together
with the use of narrow pH range Immobiline strips in
the first dimension to increase resolution should make
virtually all proteins accessible to mapping. However,
there is one major caveat, the proteome coverage that
can be achieved using 2-D gel analysis must be defined.
A detailed comparison of the mRNAs being expressed
with the level of their translation products will be necessary, in order to answer the critical question of how
much of a genome is being expressed at any given time.
This work was supported by a grant from the Swiss Federal
Institute of Technology to PJ. The authors would like to
thank John Yates and Jimmy Eng of the University of
Washington (Seattle, Washington, USA) for their Sequest
program, Matthias Mann for his Peptidesearch program,
and H . Nashimoto (Teikyo University, Utsunomiya, Japan)
for making the preliminary E. coli sequences available (the
final sequence is available on the internet at http:llbsw3.aist-nara.ac.jp).
Received September 18, 1996
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