Materials and methods
1
Reagents
If not indicated otherwise, material and chemicals were from Sigma Aldrich (St. Louis, MO) in the highest grade
available. The same Δhfq strain derived from F. tularensis LVS in earlier transcriptomic study was employed also in
this study [1].
2
Cultivation of bacteria, protein extraction and trypsin digestion
Bacteria were transferred from McLeod agar plates, inoculated into Chamberlain’s chemically defined medium [2]
and grown under mild shaking at 37 °C overnight. Next day, bacterial suspensions were prepared by diluting the
overnight cultures (100 ml, OD600 0.10–0.15) and grown under mild shaking at 37 °C. After cultures reached OD600
0.6, bacteria were harvested, washed in ice-cold PBS and resuspended in 3 ml of 8M urea in 5% sodium
deoxycholate on ice. Bacteria were lysed by passing through a chilled French pressure cell (Thermo Scientific,
Milford, USA) twice. Lysates were incubated on ice for additional 30 min, centrifuged and supernatants were filtered
through 0.22 µm syringe-driven filters. Two microliters were diluted in water 150-fold for protein concentration
assessment using microBCA Kit (Thermo Scientific, Rockford, IL). Aliquots containing 300 µg of protein were diluted
eight-fold by 200 mM triethylammonium bicarbonate buffer, pH 8.5 containing 0.1% RapiGest (Waters). Proteins
were then concentrated using Amicon Ultra filters (Millipore, Bedford, MA) with a 3 kDa molecular weight cut-off to
~50 µl. Protein concentration in the retentates was determined using BCA Kit. Proteins (80 µg) were reduced in 5mM
tris(2-carboxyethyl) phosphine hydrochloride at 37°C for 2 hours and thiol groups were blocked in 10mM methyl
methanethiosulfonate for 30 min at RT. Proteins were digested with trypsin (Promega, Madison, WI) at a 1:50
enzyme to protein ratio at 37°C overnight.
3
iTRAQ exploratory analysis
For iTRAQ proteomic analysis, four cultures (i.e. biological replicates) were prepared for both, wild type (WT) strain
and Δhfq strain.
3.1 iTRAQ labeling
Samples were vacuum-concentrated and labeled with iTRAQ 8-plex reagents (AB Sciex, Foster City, CA). Peptides
from the WT cultures were labeled with 113, 115, 117 and 119 tags, while peptides from the Δhfq cultures were
labeled with 114, 116, 118, and 121 tags. Unreacted tags were quenched by adding excess of water, samples were
vacuum-concentrated and mixed in 1:1:1:1:1:1:1:1 ratio. The mixture was acidified using TFA, spun down and the
supernatant was desalted using Oasis HLB 1cc 30 mg cartridge (Waters).
3.2 IEF fractionation, nanoLC separation and MALDI-TOF/TOF analysis of iTRAQ peptides
A portion containing ~160 µg of peptides was vacuum-dried, and redissolved in 150 µl of 4M urea, 0.5% IPG buffer,
pH 3–10, (GE Healthcare, Uppsala, Sweden) in 50% trifluoroethanol. Peptides were then applied onto a 7 cm long,
pH 3–10, linear IPG strip (GE Healthcare). IPG strip was allowed to rehydrate overnight under liquid paraffin. The
peptides were isoelectrically focused using approx. 9000 Vh, ~6h on PROTEAN IEF Cell (Bio-Rad, Hercules, USA). The
strip was washed in n-hexane and cut into fourteen equal pieces. The peptides were extracted in 200 µl of 5% AcN,
0.1% TFA for two hours. The extract from each gel piece was vacuum-dried.
Peptides were redissolved in 40 µl of mobile phase A (5% ACN, 0.1% TFA), following nanoLC peptide separation on
Ultimate 3000 (Dionex, Sunnyvale, USA). An aliquot (10-15 µl) from each IEF fraction was loaded on a µ-Precolumn
C18 PepMap, 5 µm, 100 Å, 0.3 x 5 mm (Dionex). The peptides were separated on analytical Atlantis dC18 NanoEase
column, 3 µm, 100 Å, 0.1 x 150 mm (Waters) by a linear gradient formed by mobile phase A and mobile phase B
(80% ACN, 0.1% TFA), ranging from 0–35% of mobile phase B in 62 min and from 35-50% in 18 min at a flow rate 360
nl/min. The Probot fraction collector (Dionex) collected fractions every 12 s for 75 min onto an OptiTOF LC-MALDI
plate (AB Sciex, Foster City, CA). The eluate was mixed 1:2.5 postcolumn with 3 mg/ml α-cyano-4-hydroxycinnamic
acid (LaserBioLabs, Sophia-Antipolis Cedex, France) dissolved in 70% ACN, 0.1% TFA.
MALDI analysis was performed on a 4800 MALDI-TOF/TOF Analyzer (AB Sciex). MS spectra were acquired across the
mass range of 900–3800 m/z using 750 laser shots per spectrum. A maximum of seven precursors were chosen for
fragmentation in each MS spectrum, starting with the weakest precursor. CID MS/MS spectra were acquired with a
total accumulation of 3 000 laser shots.
3.3 Data analysis
MS/MS spectra were searched in ProteinPilot 2.0.1 software (AB Sciex) using the Paragon search algorithm [3]
against the F. tularensis subsp. holarctica protein database downloaded from NCBI. The Proteomics System
Performance Evaluation Pipeline add-on to ProteinPilot was used for estimation of false positive discovery rate (FDR)
[4]. The sample was described using the following parameters: sample type - iTRAQ 8-plex (peptide labeled); Cys
alkylation – methyl methanethiosulfonate; digestion - trypsin. The processing was specified as follows: quantitate on; bias correction - on; search effort - thorough; detected protein threshold - 0.05 (10.0%). Only proteins with
FDR≤1% were further considered. Intensities of iTRAQ reporter ions were corrected using isotope correction factors
supplied with the iTRAQ kit. Only proteins with significantly altered abundance (p≤0.05) at peptide level as calculated
by ProteinPilot software in at least three of four replicates were submitted for statistical evaluation at protein level.
Data were log2 transformed and one sample t-test in GraphPad Prism 6 (GraphPad Software, La Jolla, CA) was used
to detect significantly (p≤0.05) altered proteins between strains using data from four biological replicates.
Eventually, only proteins below 10% FDR as calculated by Benjamini and Hochberg method from crude iTRAQ results,
i.e. non-filtered at peptide level, were reported.
4
Targeted LC-SRM analysis
For targeted proteomic analysis, four new bacterial cultures (i.e. biological replicates) were grown for both strains.
The cultivation and sample preparation procedures were analogous to those described above but proteins were
digested sequentially with rLys-C (Promega) in 1:100 ratio for 4 hours and then with trypsin overnight. The LC-SRM
method development, including method refinement, and final data analysis were all performed in Skyline software
(University of Washington, Seattle, WA) [5]. Tryptic peptides from four WT and four Δhfq strains were pooled to
prepare a development F. tularensis sample containing ~80 µg of peptides. This sample was used to build an MS/MS
spectra library of F. tularensis peptides and for development and refinement of the LC-SRM method. Proteins to be
analyzed using LC-SRM were selected based on the results of iTRAQ analysis.
4.1 MS/MS spectral library
Peptides were desalted using Oasis HLB 1cc 10 mg cartridge and vacuum-dried. High pH reversed-phase peptide
fractionation was performed using Alliance 2695 HPLC system (Waters). Peptides were redissolved in 20 mM NH4FA
buffer, pH 10, and injected onto a Gemini C18, 3 µm, 110 Å, 100 x 2 mm column (Phenomenex, Torrance, CA). The
peptides were eluted by an acetonitrile gradient ranging from 2% to 55% over 30 min under constant 10% flow of
200 mM NH4FA buffer, pH 10. The eluted peptides were collected between the 8th and 26th min, resulting in nine 2min fractions.
An UltiMate 3000 RSLCnano system controlled by Chromeleon software (Thermo Scientific) was used for
chromatography separation. Each fraction from high pH reversed-phase separation was vacuum dried, reconstructed
in loading phase (2% ACN, 0.05% TFA) and loaded onto a PepMap100 C18, 3 µm, 100 Å, 0.075 × 20 mm trap column
(Thermo Scientific) at 5 µl/min for 5 min. Peptides were separated on a PepMap RSLC C18, 2 µm, 100 Å, 0.075 × 150
mm analytical column (Thermo Scientific) by a gradient formed by mobile phase A (0.1% FA) and mobile phase B
(80% ACN, 0.1% FA), running from 4 to 34% in 48 min, and from 34 to 55% of mobile phase B in 15 min at a flow rate
of 0.3 µl/min at 40 °C. Eluted peptides were electrosprayed into a Q-Exactive using a Nanospray Flex source (Thermo
Scientific, Bremen, Germany). Positive ion full scan MS spectra (m/z 300-1800) were acquired using a 1×106 AGC
target in the Orbitrap at 70 000 resolution (at m/z 200). The lock mass of m/z 445.12003 ([(C2H6SiO)6+H]+) was used
for internal calibration. Top 10 precursors of charge state ≥2 and threshold intensity of 1×105 counts were selected
for HCD fragmentation, with an exclusion window of 60 s. The isolation window of 2 Da and normalized CE of 27 was
used. Each HCD spectrum was acquired at resolution 35 000, with a 5×105 AGC target and a maximum 120 ms
injection time.
HCD MS/MS spectra were searched in Proteome Discoverer software (Thermo Scientific) using MASCOT (Matrix
Science, London, UK). The tryptic specificity was set to full. The mass tolerance was set to 10 ppm for precursors and
0.02 Da for product ions. No dynamic modification was allowed while Cys thiomethylation was set as a fixed
modification. Spectra explained with a cut-off score 0.95 were extracted by Skyline software from the MASCOT .dat
file and stored in an MS/MS library. The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium [6] via the PRIDE partner repository with the dataset identifier PXD001069.
4.2 Selection of surrogate peptides and transitions
FASTA sequences were imported into Skyline software and following criteria were applied on peptide selection:
digestion – trypsin (cleavage at Arg or Lys unless Pro is the next amino acid); no missed cleavage allowed; potentially
ragged ends excluded; peptide length - between 6 and 20 amino acids; the first N-terminal amino acid excluded;
structural modifications – methylthio at Cys. Only peptides matching the criteria and present in the MS/MS library
were considered. At the transition level, the criteria were as follows: precursor charge - 2 and 3; fragment charge –
1; ion types – y and b ions; product ions ranging from (m/z>precursor)-1 to last ion; fragments within a 3 m/z
window around the precursor m/z were excluded. Both the precursor m/z and ion m/z values were restricted to
span a range from 300-1400.
4.3 Detectability of peptides in development F. tularensis sample
Six most intense product ions found in the library MS/MS spectrum that comply with the above criteria were tested.
The collision energy was calculated using default regression functions for Agilent 6490 QqQ provided in Skyline
software. Where appropriate, both doubly and triply charged precursors were tested even if MS/MS spectrum for
just one precursor was available in the library. Peptides were tested for detectability in the development F.
tularensis sample using the Agilent 1260 LC system coupled to an Agilent 6490 QqQ, controlled by MassHunter
acquisition software (Agilent, Palo Alto, CA). Peptides (4 µg) were injected onto a Poroshell 120 SB-C18, 2.7 µm, 100
× 2.1 mm column (Agilent), which was maintained at 40°C, separated using a linear gradient with constant 0.1% FA
ranging from 5% to 40% ACN over 50 min at a 300 µl/min flow rate and electrosprayed using a JetStream source. The
acquisition method involved the following parameters: drying gas flow rate of 15 l/min at 200°C, nebulizing gas flow
at 30 psi, sheath gas flow of 11 l/min at 250°C, 4000 V capillary voltage, 300 V nozzle voltage, 380 V fragmentor
voltage, 4 V cell accelerator voltage, and MS operating pressure of 5×10−5 Torr. All transitions were acquired in
positive ion mode with a dwell time of 10 ms, with both Q1 and Q3 set to unit resolution.
4.4 LC-SRM method refinement
Peptides detected as unambiguous chromatographic peaks formed by overlapping transition signals that showed
high similarity degree with peak intensities in the MS/MS spectra, were considered for final SRM method. Retention
time of these unambiguously detected peptides was used for retention time predictor. Additional peptides detected
in predicted retention time windows as unambiguous chromatographic peaks were considered as well. Peptides
containing Cys modified by thiomethyl should provide unambiguous chromatographic peaks in later retention time
than predicted. For each protein, the most intense peptide precursor was kept; nevertheless peptides without Met
and Cys, peptides without and Gln at N-terminus and peptides without Asn in a close proximity to a Gly were
strongly preferred. For these peptides, stable isotope-labeled standards (SIS) containing either 13C615N4 Arg or
3
C615N2Lys were purchased (Thermo Scientific or JPT Peptide Technologies, Berlin, Germany).
SIS peptide for EIGISICTVTR was reduced in 5 mM TCEP at 60°C for 60 min, and thiol group was blocked using MMTS
at room temperature for 10 min. The peptides were grouped into sets of four peptides, avoiding combinations of
overlapping precursor m/z, and monitored in product ion scan mode (250-1400 m/z) to obtain individual MS/MS
spectra on Agilent 6490 QqQ. Up to six most intense fragments were selected, with a focus on fragments with an
m/z higher than the precursor and including y- and b-ions, both singly and doubly charged if present. The selected
transitions were subsequently evaluated for all peptides in SRM mode, and up to three best-performing transitions
were selected per peptide. Corresponding transitions for SIS peptides were added into the final LC-SRM method.
4.5 LC-MS analysis and data evaluation
To spike SIS peptides close to the concentration of bacterial counterparts, we first performed a dilution experiment
in the development sample. The concentration of each SIS peptide closest to the ideal 1:1 ratio with the native
counterpart was selected for final analysis. For final analysis, 4 µg of peptides from each sample were injected thrice
(technical replicates). Following each injection, a standard peptide mixture and trifluoroethanol in ACN (1:1) were
each injected once to assess system stability and to wash the injection needle and loop, respectively. Three best
performing transitions were monitored per precursor. The final LC-SRM analysis was performed in the scheduled
manner. Individual peptide transitions were monitored within ±60 s window around the peptide retention time. The
total cycle time was set to 500 ms. Additional LC and MS parameters and settings were identical to those used for
the initial screening for surrogate peptides.
All peaks were manually inspected to confirm proper automatic selection and integration. All transitions free from
matrix interferences were eventually used for quantification (Supporting Information, Table S1). Total precursor
ratios to SIS were calculated as a weighted mean with SIS transition peak areas as the weights. For each sample,
precursor ratios to SIS from three analytical replicates were averaged. Subsequently, averaged precursor ratio to SIS
was normalized on sum of these values across all samples for each protein. Unpaired t-test with significance
threshold p≤0.05 was employed for statistical evaluation in GraphPad Prism 6 (GraphPad Software, La Jolla, CA).
References
[1] Meibom, K.L., Forslund, A.-L., Kuoppa, K., Alkhuder, K., et al., Hfq, a novel pleiotropic regulator of virulenceassociated genes in Francisella tularensis. Infect. Immun. 2009, 77, 1866–1880.
[2] Chamberlain, R.E., Evaluation of live tularemia vaccine prepared in a chemically defined medium. Appl Microbiol
1965, 13, 232–235.
[3] Shilov, I.V., Seymour, S.L., Patel, A.A., Loboda, A., et al., The Paragon Algorithm, a next generation search engine
that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.
Mol. Cell Proteomics 2007, 6, 1638–1655.
[4] Tang, W.H., Shilov, I.V., Seymour, S.L., Nonlinear fitting method for determining local false discovery rates from
decoy database searches. J. Proteome Res. 2008, 7, 3661–3667.
[5] MacLean, B., Tomazela, D.M., Shulman, N., Chambers, M., et al., Skyline: an open source document editor for
creating and analyzing targeted proteomics experiments. Bioinformatics 2010, 26, 966–968.
[6] Vizcaíno, J.A., Deutsch, E.W., Wang, R., Csordas, A., et al., ProteomeXchange provides globally coordinated
proteomics data submission and dissemination. Nat. Biotechnol. 2014, 32, 223–226.