Proteomic Identification of Listeria monocytogenes surface-associated proteins
Cathy XY Zhang 1,2, Marybeth C. Creskey 3, Terry D. Cyr 3, Brian Brooks 1, Hongsheng Huang
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, Franco Pagotto 2,4, Min Lin 1,2
Supporting Information
SI.1 Surface Protein Digestion of L. monocytogenes
An overnight culture of L. monocytogenes in Brain Heart Infusion (BHI) broth at 37 oC with
agitation at 225 rpm in an incubated shaker (MaxQ 4000, Barnstead) was diluted 1 in 100 in BHI
broth and subcultured. Bacterial cells were harvested when O.D.620nm reached 0.5. Each sample,
with approximately 7x 1010 cells, was washed three times in 25 ml PBS (pH 7.2). For surface
protein digestion, modified sequencing-grade trypsin (Promega, Madison, WI, USA) was added at
12.5 μg (0.5mg/ml) to approximately 7x 1010 cells in 1.5 ml of digestion buffer (20 mM TrisHCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2 and 0.6 M sucrose) and the mixture incubated for 30
min at 37 oC with 50 rpm agitation. Cells were removed by centrifugation and the supernatant
filtered through 0.22 µm centrifuge filter columns (Corning, Lowell, MA, USA). For the negative
control, cells were incubated in digestion buffer only prior to filter sterilization and subsequently
digested with 12.5 μg of trypsin. Filtrates of both samples were digested overnight at 37 oC with
an additional 2.5 µg of trypsin. Peptides were concentrated and purified using C18 spin columns
(Pierce, Rockford, IL, USA) and lyophilized with a concentrator (Oligo Prep OP120
Concentrator, Savant). Peptides were treated with 1 mM DTT (Biorad, Mississauga, Ontario,
Canada) and 1 mM iodoacetamide (Sigma, St. Louis, MO, USA) for 30 min in two consecutive
steps using 50mM ammonium bicarbonate, pH 8.0 (Sigma, St. Louis, MO, USA) as solvent and
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were subsequently concentrated and purified using C18 spin columns. Peptides were lyophilized
prior to mass spectrometry analysis.
SI.2 LC-MS/MS and MS/MS spectra Identification
For trials one and two, mass spectrometry analysis was performed on a Waters Synapt High
Definition MS (HDMS) system (Milford, MA, USA). The instrument was calibrated by infusion
prior to analysis with Glu-Fibrinopeptide (100fmol/μL). All lyophilized samples were
resuspended in 25 µl injection solvent (3% ACN, 0.2% formic acid, 0.05% TFA). For each
sample, triplicate 2 µl aliquots were analyzed by loading onto a Waters Symmetry C18 trap
column (180 µm x 20 mm with 5 µm particles) and desalting with 0.1% formic acid in water
(solvent A) for 3 min at 5.0 µl/min before separating on a Waters Nano-Acquity ultraperformance liquid chromatography (UPLC) BEH130 C18 reverse-phase analytical column (100
µm x 100 mm with 1.7 µm particles). Chromatographic separation was achieved at a flow rate of
0.5µl/min over 120 min in six linear steps as follows (solvent B was 0.1% formic acid in ACN):
initial, 3% B; 2 min, 10% B; 80 min, 30% B; 100 min, 95% B; 105 min, 95% B; 106 min, 3% B;
end, 3% B. The eluting peptides were analyzed by MS and MS/MS in data dependent acquisition
(DDA) mode. MS survey scans were 1 second in duration, and MS/MS data were collected on
the top four most abundant peaks until either the total ion count exceeded 4000 or 3 s elapsed.
All peaks selected for MS/MS analysis during the first analysis of a particular sample were used
to generate an exclusion list for the second analysis, and this was reiterated for the third analysis.
These iterative exclusion lists were generated and applied in an automated fashion using a
program called AutoCat.exe (Waters Ltd). Data were processed using the Mascot software
package (Matrix Science). The raw data were processed using Mascot Distiller (version 2.4.2.0)
to create Mascot generic files (MGFs), and the data from the triplicate injections of each sample
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were automatically combined and submitted for database searching. Combining MGFs and
queuing data for searching were carried out using Mascot Daemon (version 2.3.0), and database
searches were performed using Mascot (version 2.3), against the NCBI nr database (downloaded
on July 3rd, 2012) specifying bacteria taxonomy. A search against a decoy database was
performed to obtain an estimation of false discovery rates. Peptide and MS/MS mass tolerances
were 60 ppm and 0.05 Da, respectively, and tryptic peptides having charges from 2+ to 4+ and
up to one missed cleavages were considered. Carbamidomethylation was specified as a fixed
modification and oxidation of methionine, and deamidation of asparagine and glutamine were
specified as variable modifications. The Mascot score threshold of p<0.05 was considered
significant. Mascot identification criteria used included bold red with MudPit scoring and auto
reporting of only significant matches.
For trial three, lyophilized peptides were resuspended in 35 µl injection solvent (3% ACN, 0.2%
formic acid, 0.05% TFA in water). For each sample, triplicate 5 µl aliquots were analyzed by
loading onto a Waters TRIZAIC UPLC nanoTile (85 µm x 100 mm with 1.7 µm beads) which
integrates an analytical channel (85 µm x 100 mm with 1.7 µm beads), trapping channel (180 µm
x 20 mm with 5 µm beads) and electrospray emitter. Chromatographic separation was achieved
at a flow rate of 0.450 µl/min over 60 min in five linear steps as follows (solvent B was 0.1%
formic acid in ACN): initial, 3% B; 30 min, 40% B; 32 min, 85% B; 38 min, 85% B; 40 min, 3%
B; end, 3% B. The eluting peptides were analyzed by MS and MS/MS using a Waters Synapt
HDMS system operating in DDA mode. MS survey scans were 1 second in duration, and
MS/MS data were collected on the top four most abundant peaks until either the total ion count
exceeded 4000 or 3 s elapsed with collision energy ramping from 14 to 19 volts at low mass and
50 to 64 volts at high mass. All peaks selected for MS/MS analysis during the first analysis of a
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particular sample were used to generate an exclusion list for the second analysis, and this was
reiterated for the third analysis. These iterative exclusion lists were generated and executed in an
automated fashion using a program called AutoCat.exe developed and distributed by Waters. The
raw data were processed using Mascot Distiller (version 2.4.2.0) to create Mascot generic files
(MGFs), and the data from the triplicate injections of each sample were automatically combined
and submitted for database searching. Combining MGFs and queuing data for searching were
carried out using Mascot Daemon (version 2.3.0), and database searches were performed using
Mascot (version 2.3), against the NCBI nr database (downloaded on November 21st, 2011)
specifying bacteria taxonomy. Mascot was searched with a fragment ion mass tolerance of 0.100
Da and a parent ion tolerance of 100 ppm. Iodoacetamide derivative of cysteine was specified in
Mascot as a fixed modification. Amidation of the c-terminus, deamidation of asparagine and
glutamine and oxidation of methionine were specified in Mascot as variable modifications. The
Mascot score threshold of p<0.05 was considered significant. Mascot identification criteria used
included bold red with MudPit scoring and auto reporting of only significant matches.
SI.3 Optimizing Trypsin Digestion
Cells were grown to 0.6 O.D.620nm and washed 3 times with PBS prior to incubation in 0.15 ml of
digest buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2 and 0.6 M sucrose) at 37
o
C with agitation at 50 rpm on an incubated shaker (MaxQ 4000, Barnstead). Cells (7 x 109)
were incubated for 15, 30, 45 or 60 minutes. After incubation in digest buffer, the supernatant
was obtained, mixed with 1:1 ratio with 2x SDS-PAGE loading buffer and boiled for 5min. 30 µl
of the sample mix was loaded for SDS-PAGE and subsequent silver staining. The cell pellets
were resuspended in 1ml of 15% glycerol in BHI broth, plated at appropriate dilutions on BHI
agar plates and counted for colony forming units.
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SI. 4 Immunofluoresence of Live L. monocytogenes
Cells (3 x 108) from an overnight culture of L. monocytogenes grown in BHI at 37 oC with
agitation at 225 rpm on an incubated shaker (MaxQ 4000, Barnstead) were used for each sample.
Cell pellets were incubated in 500 μl of PBS-5% BSA for 1 hour followed by 1 hour incubation
in PBS-5% BSA with primary rabbit antibody (specific serum or pre-immunization serum) at
dilution of 1:1000. Cells were washed 2 times with 500 μl of PBS and incubated for 1 hour in
250 μl of secondary antibody conjugated with DyeLight-488 (Jackson Immunoresearch
Laboratories, West Grove, PA, USA) at dilution of 1:100. Cells were washed 3 times prior to
visualization under microscope. Exposure times for both specific serum and pre-immunization
serum were 5 milliseconds.
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