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• W1998889146 abstract "Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. Protein phosphorylation is a ubiquitous post-translational modification affecting almost every signal transduction process. Advances in speed and sensitivity of mass spectrometers, together with the higher selectivity of current phosphopeptide enrichment methods, greatly facilitated the acquisition of large scale phosphoproteomic datasets. To date, studies investigating serine, threonine and tyrosine phosphorylations in higher eukaryotes usually identify about 10,000 sites (1Lemeer S. Heck A. The phosphoproteomics data explosion.Curr. Opin. Chem. Biol. 2009; 13: 414-420Crossref PubMed Scopus (141) Google Scholar). Despite the technical advances in the phosphoproteomics research field, other types of protein phosphorylation, which have been reported to occur in a variety of organisms (2Attwood P.V. Piggott M.J. Zu X.L. Besant P.G. Focus on phosphohistidine.Amino Acids. 2007; 32: 145-156Crossref PubMed Scopus (144) Google Scholar, 3Besant P.G. Attwood P.V. Piggott M.J. Focus on phosphoarginine and phospholysine.Curr. Protein Pept. Sci. 2009; 10: 536-550Crossref PubMed Scopus (73) Google Scholar), remain scarcely studied. Examples of such noncanonical phosphoresidues are phosphoarginine, phospholysine, and (N1- or N3-) phosphohistidine, where phosphorylation occurs at the side-chain nitrogen atom (N-phosphorylation), yielding a phosphoramidate (P-N) bond that is unstable at low pH conditions. In contrast, the phosphate moiety that is covalently attached to the hydroxyl group of serine, threonine, and tyrosine (O-phosphorylation) yields a phosphoester bond that is stable at acidic pH. Of note, low pH conditions are widely employed during sample preparation for phosphoproteomic analysis, especially when phosphopeptide enrichment and separation by strong cation exchange or reverse-phase chromatography are performed. Because of the acid lability of the P-N bond, N-phosphoryl amino acids often remain unidentified even though they have been suggested to engage in important biological functions (3Besant P.G. Attwood P.V. Piggott M.J. Focus on phosphoarginine and phospholysine.Curr. Protein Pept. Sci. 2009; 10: 536-550Crossref PubMed Scopus (73) Google Scholar, 4Cieœla J. Frączyk T. Rode W. Phosphorylation of basic amino acid residues in proteins: important but easily missed.Acta Biochim. Pol. 2011; 58: 137-148PubMed Google Scholar, 5Matthews H.R. Protein kinases and phosphatases that act on histidine, lysine, or arginine residues in eukaryotic proteins: a possible regulator of the mitogen-activated protein kinase cascade.Pharmacol. Ther. 1995; 67: 323-350Crossref PubMed Scopus (158) Google Scholar). Accordingly, the general occurrence and functional relevance of N-phosphorylation are vastly unknown. So far, the phosphorylation of histidine has been described as an intermediate step in the two-component and multicomponent phosphorelay signaling pathways found in bacteria, fungi, and plants. In bacteria, the corresponding histidine kinases play a major role in perception of various stimuli, allowing their hosts to rapidly adapt to changing environmental conditions, whereas in lower eukaryotes and plants, histidine kinases appear to control more specific functions (6Wuichet K. Cantwell B. Zhulin I. Evolution and phyletic distribution of two-component signal transduction systems.Curr. Opin. Microbiol. 2010; 13: 219-225Crossref PubMed Scopus (163) Google Scholar), such as osmolarity sensing (7Maeda T. Wurgler-Murphy S.M. Saito H. A two-component system that regulates an osmosensing MAP kinase cascade in yeast.Nature. 1994; 369: 242-245Crossref PubMed Scopus (937) Google Scholar) and hormone signaling (8Nongpiur R. Soni P. Karan R. Singla-Pareek S. Pareek A. Histidine kinases in plants: cross talk between hormone and stress responses.Plant Signal. Behav. 2012; 7: 1230-1237Crossref PubMed Google Scholar), respectively. In the case of phosphoarginine, a number of studies suggested its presence in diverse organisms, as reviewed in (3Besant P.G. Attwood P.V. Piggott M.J. Focus on phosphoarginine and phospholysine.Curr. Protein Pept. Sci. 2009; 10: 536-550Crossref PubMed Scopus (73) Google Scholar). For example, Wakim and co-workers obtained mammalian cell fractions that could arginine-phosphorylate histone H3 in vitro (9Wakim B.T. Aswad G.D. Ca(2+)-calmodulin-dependent phosphorylation of arginine in histone 3 by a nuclear kinase from mouse leukemia cells.J. Biol. Chem. 1994; 269: 2722-2727Abstract Full Text PDF PubMed Google Scholar, 10Wakim B. Grutkoski P. Vaughan A. Engelmann G. Stimulation of a Ca2+-Calmodulin-activated Histone 3 Arginine Kinase in Quiescent Rat Heart Endothelial Cells Compared to Actively Dividing Cells.J. Biol. Chem. 1995; 270: 23155-23158Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), however the identity of the corresponding arginine kinase(s) was never determined. To date, the only protein arginine kinase that could be characterized in detail is the McsB protein occurring in Bacillus subtilis and closely related bacterial species (11Fuhrmann J. Schmidt A. Spiess S. Lehner A. Turgay K.R. Mechtler K. Charpentier E. Clausen T. McsB is a protein arginine kinase that phosphorylates and inhibits the heat-shock regulator CtsR.Science. 2009; 324: 1323-1327Crossref PubMed Scopus (125) Google Scholar). The McsB kinase is part of the bacterial stress response system, where it is involved in regulating CtsR, the central transcriptional repressor of class III heat shock genes. Notably, CtsR is capable of binding to DNA in a temperature dependent manner, using the beta-wing of its winged helix-turn-helix (HTH 1The abbreviations used are: GOgene ontologyHFBAheptafluorobutyric acidHTHhelix-turn-helixiTRAQisobaric tag for relative and absolute quantitationRPreverse-phaseSPEsolid-phase extractionTiO2titanium dioxide. 1The abbreviations used are: GOgene ontologyHFBAheptafluorobutyric acidHTHhelix-turn-helixiTRAQisobaric tag for relative and absolute quantitationRPreverse-phaseSPEsolid-phase extractionTiO2titanium dioxide.) as a temperature sensor. At elevated temperatures, the beta-wing induces dissociation of CtsR from DNA and consequently allowing for heat shock gene expression (12Elsholz A. Michalik S. Zühlke D. Hecker M. Gerth U. CtsR, the Gram-positive master regulator of protein quality control, feels the heat.EMBO J. 2010; 29: 3621-3629Crossref PubMed Scopus (62) Google Scholar). However, high temperature is not the only stress condition where expression of class III genes is induced. It is thus likely that McsB-mediated arginine phosphorylation is an additional mechanism inhibiting the CtsR repressor under a broad range of protein folding stress situations. Consistent with this notion, it was shown that McsB phosphorylation of CtsR inhibits binding to its operator DNA in vitro (11Fuhrmann J. Schmidt A. Spiess S. Lehner A. Turgay K.R. Mechtler K. Charpentier E. Clausen T. McsB is a protein arginine kinase that phosphorylates and inhibits the heat-shock regulator CtsR.Science. 2009; 324: 1323-1327Crossref PubMed Scopus (125) Google Scholar), that McsB and CtsR interact in vivo and that the heat shock-induced degradation of CtsR depends on the presence of McsA and McsB (13Kirstein J. Zühlke D. Gerth U. Turgay K. Hecker M. A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis.EMBO J. 2005; 24: 3435-3445Crossref PubMed Scopus (89) Google Scholar). The genes regulated by CtsR encode factors belonging to the protein quality control system, such as the ClpP protease and the associated ClpC and ClpE unfoldases, as well as regulatory proteins such as CtsR, McsA, and McsB themselves (14Derré I. Rapoport G. Msadek T. CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria.Mol. Microbiol. 1999; 31: 117-131Crossref PubMed Scopus (316) Google Scholar, 15Krüger E. Hecker M. The first gene of the Bacillus subtilis clpC operon, ctsR, encodes a negative regulator of its own operon and other class III heat shock genes.J. Bacteriol. 1998; 180: 6681-6688Crossref PubMed Google Scholar, 16Derré I. Rapoport G. Devine K. Rose M. Msadek T. ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis.Mol. Microbiol. 1999; 32: 581-593Crossref PubMed Scopus (90) Google Scholar). ClpCP forms an ATP-dependent protease complex, similar to the eukaryotic proteasome, that not only carries out protein quality control, by removing unfolded proteins (17Krüger E. Witt E. Ohlmeier S. Hanschke R. Hecker M. The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins.J. Bacteriol. 2000; 182: 3259-3265Crossref PubMed Scopus (131) Google Scholar, 18Schlothauer T. Mogk A. Dougan D. Bukau B. Turgay K.R. MecA, an adaptor protein necessary for ClpC chaperone activity.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2306-2311Crossref PubMed Scopus (117) Google Scholar), but also functions as a regulatory protease in developmental processes such as sporulation (19Pan Q. Losick R. Unique degradation signal for ClpCP in Bacillus subtilis.J. Bacteriol. 2003; 185: 5275-5278Crossref PubMed Scopus (29) Google Scholar) and competence (20Turgay K. Hahn J. Burghoorn J. Dubnau D. Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor.EMBO J. 1998; 17: 6730-6738Crossref PubMed Google Scholar). In addition, ClpCP itself appears to function as a central regulator in the bacterial stress response, by mediating CtsR degradation upon heat shock (21Krüger E. Zühlke D. Witt E. Ludwig H. Hecker M. Clp-mediated proteolysis in Gram-positive bacteria is autoregulated by the stability of a repressor.EMBO J. 2001; 20: 852-863Crossref PubMed Scopus (115) Google Scholar, 22Kirstein J. Dougan D. Gerth U. Hecker M. Turgay K.R. The tyrosine kinase McsB is a regulated adaptor protein for ClpCP.EMBO J. 2007; 26: 2061-2070Crossref PubMed Scopus (68) Google Scholar) and inhibiting the activity of McsB, according to in vitro assays (13Kirstein J. Zühlke D. Gerth U. Turgay K. Hecker M. A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis.EMBO J. 2005; 24: 3435-3445Crossref PubMed Scopus (89) Google Scholar). Therefore, the activation of the stress response in B. subtilis is regulated by an intricate feedback loop resulting from the concerted activities of CtsR, McsB and ClpCP. gene ontology heptafluorobutyric acid helix-turn-helix isobaric tag for relative and absolute quantitation reverse-phase solid-phase extraction titanium dioxide. gene ontology heptafluorobutyric acid helix-turn-helix isobaric tag for relative and absolute quantitation reverse-phase solid-phase extraction titanium dioxide. A further regulatory level is represented by the YwlE phosphatase that counteracts the activity of the McsB arginine kinase (13Kirstein J. Zühlke D. Gerth U. Turgay K. Hecker M. A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis.EMBO J. 2005; 24: 3435-3445Crossref PubMed Scopus (89) Google Scholar). Based on sequence homology, YwlE was initially annotated as a tyrosine phosphatase. However, in vitro assays using phosphopeptide substrates demonstrated that, even though phosphotyrosine phosphatase activity can occur at pH 5 (23Musumeci L. Bongiorni C. Tautz L. Edwards R. Osterman A. Perego M. Mustelin T. Bottini N. Low-molecular-weight protein tyrosine phosphatases of Bacillus subtilis.J. Bacteriol. 2005; 187: 4945-4956Crossref PubMed Scopus (45) Google Scholar), at physiological pH YwlE activity is highly specific toward phosphoarginine (24Fuhrmann J. Mierzwa B. Trentini D.B. Spiess S. Lehner A. Charpentier E. Clausen T. Structural Basis for Recognizing Phosphoarginine and Evolving Residue-Specific Protein Phosphatases in Gram-Positive Bacteria.Cell Rep. 2013; 3: 1832-1839Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). By systematically analyzing phosphatase-deficient B. subtilis strains, the latter study also revealed that YwlE is the only arginine phosphatase in this bacterium. In sum, these findings indicate that arginine phosphorylation is a carefully balanced post-translational modification resulting from the antagonistic activity of very specific protein kinases and phosphatases (24Fuhrmann J. Mierzwa B. Trentini D.B. Spiess S. Lehner A. Charpentier E. Clausen T. Structural Basis for Recognizing Phosphoarginine and Evolving Residue-Specific Protein Phosphatases in Gram-Positive Bacteria.Cell Rep. 2013; 3: 1832-1839Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). To better understand how McsB-mediated arginine phosphorylation affects the bacterial stress response and to identify specific targets of McsB, we studied changes in the B. subtilis arginine phosphoproteome under different environmental conditions. For this purpose, we developed a quantitative MS approach adapted to the special chemical properties of phosphoarginine. Quantitative data uncovered major physiological McsB targets, which comprise the main regulators and executors of the bacterial stress response system including CtsR, HrcA, GroEL, ClpC and ClpP. Additionally, the obtained results demonstrate that the improved phosphoproteomic method reported here will be of great value for the scientific community in investigating the prevalence and the biological function of acid-labile phosphorylations, that so far escaped systematic analysis. All model peptides for acidic hydrolysis were synthesized in house using FMOC-based coupling chemistry. Peptides were HPLC purified and identity and purity were validated by MALDI mass spectrometry. Arginine phosphorylated peptides were synthesized without the phosphomoiety. Phosphorylation was subsequently introduced in vitro using purified recombinant McsB arginine kinase from Geobacillus stearothermophilus. The resulting peptide mixture was again HPLC fractionated (Merck, Germany) on a semipreparative C18 column (GeminiC18, 15 cm × 2 mm × 5 μm, Phenomenex, Germany) using an ammonium bicarbonate buffer at pH 7 to obtain 99% pure arginine phosphorylated peptide. Aliquots of 20 pMol/reaction of the phosphopeptide ac-K(pR)GGGGYIKIIKV were diluted in 200 mm phosphate buffer to a final concentration of 0.1 μm. All hydrolysis reactions were carried out in 200 μl scale. Time points were taken after 5, 15, 30, 60, 120, and 240 min of reaction time by neutralizing the reaction mixture with 1 m HEPES, pH 7.2. For the first replicate, samples of each time point were injected on an Ultimate plus HPLC system for C18-RP separation (PepMapAcclaim, 15 cm × 75 μm × 3 μm, 100Å, Dionex-Thermo-Fisher Scientific, United States) and detected on a LCQ DecaXP mass spectrometer (Thermo-Fisher Scientific) in data dependent mode. The second replicate was analyzed on an Ultimate 3000 beta HPLC system coupled to an LTQ FT hybrid mass spectrometer (Dionex-Thermo-Fisher Scientific) applying the same separation conditions. Mass traces corresponding to the 2+ and 3+ charge states of phosphorylated and hydrolyzed peptide were manually integrated to determine the abundance of each peptide form at the respective incubation time. The ywlE-deficient mutant was generated by replacing the ywlE gene with a spectinomycin resistance cassette carrying a transcriptional terminator. DNA fragments corresponding to the chromosomal DNA regions located immediately upstream and downstream of the ywlE coding sequence were generated by PCR using oligonucleotide pairs ywlE_fw_UP (5′-GCCGCCGGAGGAAGAGTGAT-3′) and ywlE_rev_UP_spec (5′-CGCTCACGAAGGGATTCGATAGACAAAAATAATATCCAT-3′); ywlE_fw_DW_spec (5′-GTTCCTTCGATAGTTTATTATTGTCAGAAAATCTGCAAAC-3′) and ywlE_rev_DW (5′-TAAACAGCACCCCTACAGGT-3′); A DNA fragment carrying the spectinomycin resistance cassette was generated using the primers reverse complementary to ywlE_rev_UP_spec and ywlE_fw_DW_spec. The three PCR fragments were ligated by PCR and then introduced into competent B. subtilis 168 [ATCC 2385] cells as described previously (25Anagnostopoulos C. Spizizen J. Requirements for transformation in Bacillus subtilis.J. Bacteriol. 1961; 81: 741-746Crossref PubMed Google Scholar). Spectinomycin-resistant mutants that resulted from double cross-over events in which 421 bp of the ywlE coding sequence was replaced by the spectinomycin resistance cassette were selected on LB agar plates containing spectinomycin (200 μg/ml). The ΔywlEΔmcsB double mutant was generated by introducing chromosomal DNA prepared from B. subtilis ΔmcsB (26Kobayashi K. Ehrlich S.D. Albertini A. Amati G. Andersen K.K. Arnaud M. Asai K. Ashikaga S. Aymerich S. Bessieres P. Boland F. Brignell S.C. Bron S. Bunai K. Chapuis J. Christiansen L.C. Danchin A. Débarbouillé M. Dervyn E. Deuerling E. Devine K. Devine S.K. Dreesen O. Errington J. Fillinger S. Foster S.J. Fujita Y. Galizzi A. Gardan R. Eschevins C. Fukushima T. Haga K. Harwood C.R. Hecker M. Hosoya D. Hullo M.F. Kakeshita H. Karamata D. Kasahara Y. Kawamura F. Koga K. Koski P. Kuwana R. Imamura D. Ishimaru M. Ishikawa S. Ishio I. Le Coq D. Masson A. Mauëlx C. Meima R. Mellado R.P. Moir A. Moriya S. Nagakawa E. Nanamiya H. Nakai S. Nygaard P. Ogura M. Ohanan T. O'Reilly M. O'Rourke M. Pragai Z. Pooley H.M. Rapoport G. Rawlins J.P. Rivas L.A. Rivolta C. Sadaie A. Sadaie Y. Sarvas M. Sato T. Saxild H.H. Scanlan E. Schumann W. Seegers J.F.M.L. Sekiguchi J. Sekowska A. Séror S.J. Simon M. Stragier P. Studer R. Takamatsu H. Tanaka T. Takeuchi M. Thomaides H.B. Vagner V. van Dijl J.M. Watabe K. Wipat A. Yamamoto H. Yamamoto M. Yamamoto Y. Yamane K. Yata K. Yoshida K. Yoshikawa H. Zuber U. Ogasawara N. Essential Bacillus subtilis genes.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 4678-4683Crossref PubMed Scopus (1143) Google Scholar) into competent B. subtilis ΔywlE cells. Mutants were selected on LB agar plates containing spectinomycin, erythromycin (5 μg/ml), and lincomycin (20 μg/ml). The gene replacements were confirmed by PCR and sequencing analysis. B. subtilis cultures were grown in liquid Luria-Bertani (LB) medium. For the ΔywlE strain, spectinomycin (200 μg/ml) was added to the media, whereas for the ΔywlEΔmcsB strain both spectinomycin and erythromycin (5 μg/ml) were used. For phosphoproteomic experiments, cultures grown overnight at 37 °C were diluted in 200 ml of fresh medium for each condition to an A600 of 0.05 and grown at 37 °C under orbital shaking until an A600 of 0.6 was reached. For heat stress, the culture flask was transferred to a 50 °C water bath for 20 min under orbital shaking. Oxidative stress was induced by adding 1 mm diamide. The control culture remained at the initial growth conditions. After the 20 min incubation time all cells were harvested by centrifugation at 4 °C. Obtained pellets were washed once with ice cold PBS buffer and stored at −80 °C until further use. Peroxidated vanadate species have been described as potent tyrosine phosphatase inhibitors (27Pumiglia K.M. Lau L.F. Huang C.K. Burroughs S. Feinstein M.B. Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide).Biochem. J. 1992; (Pt 2): 441-449Crossref PubMed Scopus (102) Google Scholar). Because YwlE are structurally and mechanistically related to low molecular weight tyrosine phosphatases, we reasoned that this inhibitor might also eliminate YwlE activity. Sodium pervanadate was prepared from sodium ortho-vanadate and H2O2. Briefly, 500 mm sodium ortho-vanadate in water was brought to pH 7 with concentrated HCl and boiling until the yellow color disappeared. After dilution to 10 mm concentration in 100 mm HEPES pH 7.5, 3% H2O2 in 100 mm HEPES was added and the solution was incubated at room temperature for 5 min. To remove residual H2O2, 1 μl of 1 mg/ml bovine catalase was added. The resulting pervanadate solution was added to the cell medium in a final concentration of 0.4 mm 5 min before the cells were exposed to heat shock conditions at 50 °C for 20 min. Controls in which the cells were not exposed to higher temperature were performed in parallel. The pellet of a 200 ml cell culture (approx. 1 g) grown to an OD600 of 0.6 was transferred into freezer mill sample vials under liquid nitrogen. The cells were crushed in a freezer mill (Spex, New Jersey) applying the following settings: 10 cycles consisting of 2 mins of grinding (15 counts/second) followed by 3 mins of cooling per cycle. The obtained cell powder was stored at −80 °C until further use. Samples were allowed to warm until a suspension was formed. This was transferred into 1.5 ml Eppendorf vials and one sample volume of lysis buffer (100 mm Tris, pH 7.5, 4% SDS, 200 mm dithiotreitol, 3 mm of each phosphatase inhibitor: sodium vanadate, potassium fluoride, sodium pyrophosphate, and glycerol phosphate) was added. Samples were homogenized with a sonication probe (UPH100 0.5 mm, Hielscher, Germany) to dissolve proteins and shear DNA. After centrifugation, the supernatant was incubated at 56 °C for 50 min to reduce disulfide bonds. Immediately after reduction, the protein solution was diluted with 12 ml 100 mm Tris (pH 7.5, containing 8 m Urea and 25 mm iodoacetamide) in an Amicon filter unit (Millipore, United States, MWCO 30000) for alkylation of free cysteines. After incubation for 45 min in the dark, samples were washed according to the FASP protocol (28Wis′niewski J. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5099) Google Scholar) by centrifugation at 4000 g applying 2 × 15 ml 100 mm Tris (pH 7.5, containing 8 m Urea), 2 × 15 ml 100 mm Tris (pH 7.5, containing 4 m Urea), and 2 × 100 mm ammonium bicarbonate. The retentate was reduced to 2–3 ml in the last washing step. The protein content was determined after the last 4 m urea wash using a Bradford Protein Assay (Bio-Rad, Germany). Lyophilized Trypsin Gold (Promega, Madison, WI) was allowed to heat to room temperature before it was dissolved to 1 μg/μl with 100 mm ammonium bicarbonate solution. Tryptic digest of all B. subtilis whole cell lysates was achieved with a protein to trypsin ratio of 100:1 for 14 h at 37 °C in the filter devices. The level of digestion was monitored by retention time and UV intensity (214 nm) distribution on RP-HPLC separation of 0.25 to 0.5 μg protein on a monolithic column (Ultimate Plus equipped with a PepSwift PS-DVB column, 5 cm x 200 μm, all Dionex-Thermo-Fisher Scientific). Further, the peptide content was determined via absorbance at 280 nm in a NanoDrop device (Thermo-Fisher Scientific). To accomplish a high degree of iTRAQ labeling, 0.5 mg of peptides from each sample were purified from ammonium bicarbonate and other reagents from the sample preparation by RP-C18 solid-phase extraction (SPE) at neutral pH (Strata-X 200 mg, 6 ml cartridge, Phenomenex, Germany). The eluent was dried under vacuum and re-dissolved in 50 μl 100 mm TEAB buffer. ITRAQ reagents (Invitrogen, New York) were dissolved in 150 μl ethanol, added to the peptide solution, and the mixture was incubated for 4h at room temperature. Labeling efficiency was monitored by LC-MS/MS analysis of each individual labeling reaction and modification of N termini and lysines of more than 97% were considered as completely labeled, otherwise fresh iTRAQ reagent was applied. Furthermore, to confirm an acid-labile arginine phosphorylation as PTM, two aliquots were prepared for the control of the first biological replicate, one was labeled with iTRAQ reagent 114 and the other one with reagent 117. The aliquot with the 114-label was incubated in 1% TFA at 60 °C for 1 h to hydrolyze all N-phosphorylations before mixing with the other aliquots. In the second replicate this treatment was omitted, because ETD fragmentation allowed sufficient phosphosite localization confidence. For quantitative protein and phosphorylation analysis, individual samples (unstressed control, acid-treated unstressed control, heat shock, and oxidative stress) were mixed in a 1:1:1:1 ratio according to their protein amount. The excess of quenched labeling reagent over peptides was removed by a second solid-phase extraction step on a 6 ml C18 cartridge Strata-X after mixing the 4 iTRAQ channels. Apart from the regulation of phosphorylation sites, also alterations in the protein abundance are important to reveal cellular processes occurring during heat shock or oxidative stress exposure. Therefore, an aliquot of 100 μg of the final mixture was fractionated by strong cation exchange chromatography on a 1 mm Polysulfoethyl column. Solvents for the SCX were 5 mm sodium phosphate, pH 2.7 for buffers A and B and pH 6 for buffer C, respectively. For efficient elution, solvent B also contained 1 m sodium chloride. Peptides were bound to the resin in 100% solvent A. Peptide separation was achieved by a linear gradient combining increasing percentages for buffer B and C over 45 min. Fractions were collected every 5 min and analyzed by LC-MS/MS as described below. The iTRAQ-labeled peptide mixture containing 0.25 mg sample per channel was lyophilized and re-dissolved in 150 μl TiO2 loading buffer (300 mg/ml lactic acid, 12.5% acetic acid (AcOH), 0.2% heptafluorobutyric acid (HFBA), 60% acetonitrile (ACN), pH 4 with NH3) immediately before incubation with the TiO2 resin (Titansphere, 5 μm, GL Sciences, Japan). After incubation for 35 min at 20 °C, unbound peptides were removed by filter centrifugation in Mobicol devices (MoBiTec, Germany) and the resin was washed with additional 150 μl of loading buffer. Further removal of unphosphorylated peptides was achieved by subsequent washing with 400 μl of solvent A (200 mg/ml lactic acid, 75% ACN, 2% trifluoroacetic acid (TFA), 2% HFBA), solvent B (200 mg/ml lactic acid, 75% ACN, 10% AcOH, 0.1% HFBA, pH 4 with NH3), and solvent C (80% ACN, 10% AcOH). The TiO2 resin was re-suspended in each washing step. For elution of phosphopeptides, the resin was incubated twice with 100 μl of 1% NH3 solution containing 30 mm ammonium phosphate for 15 min. The sample volume was reduced under low pressure and a pH of 7 was adjusted before injection onto the LC column. RP separation of all peptide mixtures was achieved on an Ultimate 3000 RSLC nano-flow chromatography system (Thermo-Fisher). An alternative loading solvent to 0.1% TFA was required, because already short washing periods of arginine-phosphorylated samples under these conditions lead to significant hydrolysis of the acid-labile phosphoarginine. Therefore, 0.5% AcOH (pH 4.5 with NH3) at a flow rate of 15 μl/min was used to remove salts after loading the peptide mixture onto the precolumn (PepMapAcclaim C18, 2 cm × 0.1 mm, 5 μm, Dionex-Thermo-Fisher). Peptide separation was achieved on a C18 separation column (PepMapAcclaim C18, 50 cm × 0.75 mm, 2 μm, Dionex-Thermo-Fisher) by applying a linear gradient from 2% to 40% solvent B (80% ACN, 0.1% FA) in 180 or 240 min. Solvent A was 2% ACN, 0.1% FA. Under these conditions, the hydrolysis of the phosphoarginine mark was estimated to be very low, ranging from 5 to 10%. The separation was monitored by UV detection and the outlet of the detector was directly coupled to the nano-Electrospray ionization source (Proxeon Biosystems, Denmark) for MS analysis. The sample was infused into the LTQ Orbitrap Velos ETD mass spectrometer (Thermo-Fisher" @default.
• W1998889146 created "2016-06-24" @default.
• W1998889146 creator A5014342884 @default.
• W1998889146 creator A5046572854 @default.
• W1998889146 creator A5046833624 @default.
• W1998889146 creator A5055830366 @default.
• W1998889146 creator A5063181496 @default.
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• W1998889146 date "2014-02-01" @default.
• W1998889146 modified "2023-10-16" @default.
• W1998889146 title "Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response" @default.
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