Biology Reference
In-Depth Information
Proteomics of Model Bacterial Communities
High density bacterial communities were generated by the method of Merritt et al.
[44]. Bacteria were cultured to mid-log phase, harvested by centrifugation and resus-
pended in pre-reduced PBS (rPBS). 1 × 10
9
cells of
P. gingivalis
were mixed with an
equal number of
S. gordonii
and
F. nucleatum
as a combination of the three species.
Porphyromonas gingivalis
cells alone were also used as a control. Two independent
biological replicates from separate experiments comprised of at least two technical
replicates were analyzed. Bacteria were centrifuged at 3,000 g for 5 min, and pel-
lets were held in 1 ml rPBS in an anaerobic chamber at 37°C for 18 hr. The bacte-
rial cells remain viable under these conditions, as determined by both colony counts
and live/dead fluorescent staining. Supernatant and bacterial cells were separated and
processed separately. Bacterial cells were lyzed with ice cold sterile distilled water
and proteins were digested with trypsin as previously described for
P. gingivalis
[33],
then fractionated on a 2.0 mm × 150 mm YMC polymer C18 column. There were
five pre-fractions collected for each cellular sample, with a final volume of 50 μl
for each fraction. The 2D capillary High Performance Liquid Chromatography/ Mass
Spectrometry/Mass Spectrometry (HPLC/MS/MS) analyses [32, 45, 46] were con-
ducted using an in-house fabricated semi-automated system, consisting of a Thermo
LTQ mass spectrometer (Thermo Fisher Corp. San Jose, CA, USA), a Magic 2002
HPLC (Michrom BioResouces, Inc., Auburn, CA, USA), a Pump 11 Plus syringe pump
(Harvard Apparatus, Inc., Holliston, MA, USA), an Alcott 718 autosampler (Alcott
Chromatography, Inc., Norcross, GA, USA) and a micro-electrospray interface built
in-house. About 2 μl of sample solution was loaded into a 75 μm i.d. × 360 μm o.d.
capillary column packed with 11 cm of AQUA C18 (5 μm, Phenomenex, Torrance,
CA, USA) and 4 cm of polysulfoethyl aspartamide SCX (strong cation exchange) resin
(PSEA, 5 μm, Michrom BioResouces, Inc.). The peptides were eluted with a seven step
salt gradient (0, 10, 25, 50, 100, 250, and 500 mM ammonium acetate) followed by
an acetonitrile gradient elution (Solvent A: 99.5% water, 0.5% acetic acid. Solvent B:
99.5% acetonitrile, 0.5% acetic acid), 5% B hold 13 min, 5-16% B in 1 min, hold 6
min, 16-45% B in 45 min, 40-80% B in 1 min, hold 9 min, 80-5% B in 5 min, then
hold 10 min. For the secreted proteins in the supernatant no pre-fractionation or SCX
was performed, and 4 μl of digested sample was loaded into a 75 μm i.d. × 360 μm
o.d. column packed with 11 cm AQUA C18 for a single dimension of capillary HPLC/
tandem MS analysis. After 20 min of flushing with 5% acetonitrile, peptides were
eluted by an acetonitrile gradient (5-12% B in 1 min, hold 9 min, 12-40% B in 50 min,
40-80% B in 1 min, hold 10 min, 80-5% B in 5 min, hold 14 min). The MS
1
(First stage
of tandem mass spectrometry) scan range for all samples was 400-2,000 m/z. Each MS
1
scan was followed by 10 MS
2
(Second stage of tandem mass spectrometry) scans in a
data dependent manner for the 10 most intense ions in the MS
1
scan. Default parameters
under Xcalibur 1.4 data acquisition software (Thermo Fisher) were used, with the ex-
ception of an isolation width of 3.0 m/z units and a normalized collision energy of 40%.
Data Processing and Protein Identification
Raw data were searched by SEQUEST [34] against a FASTA protein ORF database
consisting of the Ver. 3.1 curation of
P. gingivalis
W83 (2006, TIGR-CMR [47]),
S.
