Macek, B., Mijakovic, I., Olsen, J.V., Gnad, F., Kumar, C., Jensen, P.R. and Mann, M. (2007) The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis. Mol Cell Proteomics [Epub ahead of print]
Cell culture, fractionation and peptide preparation
Wild type B. subtilis cells (strain 168) were grown in 12 liters of LB medium under vigorous shaking. At OD600=1, the cells were precipitated by centrifugation (5 minutes at 600 g) and resuspended in lysis buffer containing 50 mM Tris-Cl (pH 7.5), 5 mg/ml lysozyme, and 5 mM of each of the following phosphatase inhibitors: sodium fluoride, 2-glycerol phosphate, sodium vanadate and sodium pyrophosphate (Sigma). Cell wall lysis was performed for 15 minutes at 37° C and cell membranes were disrupted by sonication. Dnase I (100 µg/ml) (Sigma) was added to the lysate and incubated for additional 10 minutes at 37° C. N-octylglucoside detergent (Sigma) was added to final concentration of 1% for more efficient extraction and solubilization of membrane proteins. Cellular debris was removed by centrifugation at 25,000x g for 30 minutes. The crude protein extract was extensively dialyzed against deionized water and finally lyophilized.
Protein digestion and phosphopeptide enrichment
Protein concentration in the crude protein extract was estimated by Bradford reagent (Bio Rad). About 10 mg of protein extract was dissolved and denatured in 6 M urea and 2 M thiourea, reduced with 1 mM dithiothreitol (DTT) for 45 minutes at room temperature (RT) and carbamidomethylated with 5 mM iodoacetamide for 45 minutes at RT in the dark. Alkylated proteins were digested first with endopeptidase Lys-C (Waco) for 3 hours, after which the solution was diluted four times with deionized water, and then further digested with sequencing grade modified trypsin (Promega) overnight. Protease/protein ratio was in both cases 1/50. Resulting peptide mixture was acidified with trifluoroacetic acid (TFA) to pH<3 and subjected to two stages of phosphopeptide enrichment. In the first stage, strong cation exchange chromatography was performed as described previously (1), with minor modifications. Samples were loaded onto a 1 mL Resource S column (GE Healthcare), in solvent A (5 mM KH2PO4, 30% acetonitrile, 0.1% trifluoroacetic acid, pH=2.7), at a flow rate of 1 mL/min. Elution was performed with a gradient of 0-30% solvent B (5 mM KH2PO4, 30% acetonitrile, 350 mM KCl, 0.1% trifluoroacetic acid, pH=2.7), over 30 minutes. Fifteen 2-mL fractions, as well as the flow-through were collected and subjected separately to the second stage of phosphopeptide enrichment. The second stage of phosphopeptide enrichment was performed using TiO2 chromatography (2;3). The TiO2 beads (kindly provided by GL Sciences, Japan), and all SCX fractions were pre-incubated with 5 mg/mL 2,5-dihydroxybenzoic acid in 80% acetonitrile. Each SCX fraction was then added to a 2 mL reaction tube containing ~10 mg of the TiO2 beads and incubated batch-wise with end-over-end rotation for 30 minutes. After incubation, the beads were spun down and washed 2 times with acetonitrile/water (1:1) solution containing 0.2% trifluoroacetic acid. Bound peptides were eluted from the column with 0.5% ammonium solution, pH 10.5, in 40% acetonitrile, dried almost to completeness and reconstituted in 1% TFA, 2% acetonitrile in water for LC-MS analysis.
Liquid chromatography - mass spectrometry
Liquid chromatography was performed on a 1100 nano-HPLC system (Agilent Technologies), fitted with an in-house made 75 µm reverse phase C18 column, as described previously (4). Each sample was loaded in solvent A (0.5% acetic acid in water) and eluted with a segmented gradient of 10-60 % solvent B (80% acetonitrile, 0.5% acetic acid in water) over 120 minutes. The HPLC was coupled to either LTQ-FT, or LTQ-Orbitrap mass spectrometer (Thermo Electron), via a nano-LC interface (Proxeon Biosystems). In the LTQ-FT mass spectrometer samples were measured in duplicate; in the first measurement the survey scan was performed in the FT-ICR analyzer and followed by SIM scans of the three most intense peptide ions at resolution (R) of 50,000, and their MS2 fragmentation in the linear ion trap (LTQ) ("FT-SIM" method) (5). The second LTQ-FT measurement was performed by the survey scan acquisition in the FT-ICR (at R=100,000), MS2 of the five most intense peptide ions in the LTQ and MS3 of all ions showing the neutral loss of phosphoric acid (98 Da) from the precursor ion, as described (1) ("FT-Top5" method). In the LTQ-Orbitrap mass spectrometer samples were measured in triplicate. In the first measurement the survey scan was performed in the orbitrap analyzer (at R=60,000) and was followed by MS2 of the three most intense ions in both the orbitrap (at R=15,000) and the LTQ analyzers ("Orbitrap-FT" method). The second measurement was performed by the survey scan acquisition in the orbitrap (at R=30,000) and subsequent MS2 in both the C-trap (acquired in the orbitrap at R=15,000), and the LTQ ("Orbitrap HCD" method). The final measurement was performed by the survey scan acquisition in the orbitrap (at R=60,000) and MS2 of the five most intense ions in the LTQ ("Orbitrap-Top5" method). All measurements in the orbitrap mass analyzer were performed with on-the-fly internal recalibration by "locking" to polydimethylcyclosiloxane ions at m/z 445.120025 and 429.088735, as described previously (6), for further improvement of mass accuracy.
Data processing, validation and analysis
Peak lists for database search were produced in the Mascot generic format using the in-house developed software DTASuperCharge for the LTQ-FT spectra and Raw2msm (6) for the LTQ-Orbitrap spectra. Since bacteria were grown on the LB medium containing yeast extract, a concatenated database consisting of forward and reversed sequences of the Bacillus subtilis strain 168 (forward protein database downloaded from TIGR), Saccharomyces cereviseae (yeast_orf database, Saccharomyces genome database, Stanford University) and 26 most commonly observed contaminants in MS measurements was created and searched using the Mascot search engine (Matrix science). The search criteria were as follows: full tryptic specificity was required; 2 missed cleavages were allowed; carbamidomethylation was set as fixed modification; oxidation (M), N-acetylation (protein), phosphorylation (STY), (H) and (D) were set as variable modifications; precursor ion mass tolerances were 10 ppm for all measurements; fragment ion mass tolerance was 0.5 Da for all MS2 spectra acquired in the LTQ and 0.02 Da for all MS2 spectra acquired in the orbitrap mass analyzer. Mass spectra of identified phosphopeptides were analyzed using the MSQuant software. All spectra were manually validated and the following acceptance criteria were applied: precursor ion mass tolerance required for all orbitrap and "FT-SIM" measurements was 5 ppm, and for "FT-Top5" was 10 ppm. All P-Ser and P-Thr peptides were required to show a pronounced neutral loss of phosphoric acid from the precursor ion and/or fragment ions, or trigger the neutral loss-dependant MS3 scan. Only peptides containing more than 7 amino acid residues were considered and extensive coverage of b- and/or y-ion series was required. All proline-containing peptides were required to show pronounced cleavage N-terminally to the Pro residue. Phosphopeptides that did not show this typical fragmentation pattern were accepted only if they were detected in two or more different measurements, or in two or more forms (e.g. with or without methionine oxidation, or as a full and a missed cleavage). In phosphopeptides with multiple potential phosphorylation sites, the probabilities for phosphorylation at each site were calculated from the PTM scores, as described previously (3).
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4. Olsen, J. V., Ong, S. E., and Mann, M. (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics 3, 608-14.
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