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Citrullinome of Porphyromonas gingivalis Outer Membrane Vesicles: Confident Identification of Citrullinated Peptides

2019; Elsevier BV; Volume: 19; Issue: 1 Linguagem: Inglês

10.1074/mcp.ra119.001700

1535-9484

Daniel Nyberg Larsen, Christian Engelbrecht Mikkelsen, Mads Kierkegaard, Grzegorz Bereta, Zuzanna Nowakowska, Jakub Zbigniew Kaczmarek, Jan Potempa, Peter Højrup,

Peptidase Inhibition and Analysis

Porphyromonas gingivalis is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, Citrullia, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification. Porphyromonas gingivalis is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, Citrullia, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification. Citrullination is a deimination of arginine, which results in the loss of a single nitrogen and hydrogen along with the addition of an oxygen, resulting in a mass shift of 0.984 Da and loss of a single charge. Citrullination is a post-translational modification that can only occur on arginine residues, either on the N- or C-terminal of the peptides or internally. Citrullination occurs in physiological and pathological conditions and is thought to play a range of different functions. The human peptidyl arginine deiminases (PAD) 1The abbreviations used are:PADhumane peptidyl arginine deiminaseOMVouter membrane vesiclesWTP. gingivalis W83 wild-typeΔPPADW83 knock-out mutant of peptidyl arginine deiminaseC351AW83 expressing PPAD with the active site cysteine mutated to alanine2Dtwo-dimensional HFBA-basedLC-MSliquid chromatography mass spectrometryPPADbacterial P. gingivalis peptidyl arginine deiminaseRArheumatoid arthritisPGE2prostaglandin E2ACPAanti-citrullinated protein antibodiesRgpA/BArg-specific gingipainsMSmass spectrometryHFBAheptafluorobutyric acidFAformic acidHPLChigh performance liquid chromatographyAAAamino acid analysismgfMascot Generic File formatmgxMascot Generic eXtendedRTretention timeTICtotal ion countT9SStype IX secretion system. 1The abbreviations used are:PADhumane peptidyl arginine deiminaseOMVouter membrane vesiclesWTP. gingivalis W83 wild-typeΔPPADW83 knock-out mutant of peptidyl arginine deiminaseC351AW83 expressing PPAD with the active site cysteine mutated to alanine2Dtwo-dimensional HFBA-basedLC-MSliquid chromatography mass spectrometryPPADbacterial P. gingivalis peptidyl arginine deiminaseRArheumatoid arthritisPGE2prostaglandin E2ACPAanti-citrullinated protein antibodiesRgpA/BArg-specific gingipainsMSmass spectrometryHFBAheptafluorobutyric acidFAformic acidHPLChigh performance liquid chromatographyAAAamino acid analysismgfMascot Generic File formatmgxMascot Generic eXtendedRTretention timeTICtotal ion countT9SStype IX secretion system. 1, 2, 3, 4, and 6 exert different roles as a result of expression in different cellular environments. PAD1 citrullinates keratin and filaggrin, which is important for terminal differentiation of keratinocytes (1Nachat R. Méchin M.C. Takahara H. Chavanas S. Charveron M. Serre G. Simon M. Peptidylarginine deiminase isoforms 1–3 are expressed in the epidermis and involved in the deimination of K1 and filaggrin.J. Invest. Dermatol. 2005; 124: 384-393Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 2Ishida-Yamamoto A. Senshu T. Eady R.A.J. Takahashi H. Shimizu H. Akiyama M. Iizuka H. Sequential reorganization of cornified cell keratin filaments involving filaggrin-mediated compaction and keratin 1 deimination.J. Invest. Dermatol. 2002; 118: 282-287Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). PAD2 citrullinates myelin basic protein, which is involved in the myelin sheath formation (3Lamensa J.W.E. Moscarello M.A. Deimination of Human Myelin Basic Protein by a Peptidylarginine Deiminase from Bovine Brain.J. Neurochem. 1993; 61: 987-996Crossref PubMed Scopus (99) Google Scholar, 4Finch P.R. Wood D.D. Moscarello M.A. The presence of citrulline in a myelin protein fraction.FEBS Lett. 1971; 15: 145-148Crossref PubMed Scopus (40) Google Scholar). PAD3 is involved in hair growth by citrullination of trichohyalin (5Steinert P.M. Parry D.A.D. Marekov L.N. Trichohyalin mechanically strengthens the hair follicle: Multiple cross-bridging roles in the inner root sheath.J. Biol. Chem. 2003; 278: 41409-41419Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 6Tarcsa E. Marekov L.N. Mei G. Melino G. Lee S.-C. Steinert P.M. Protein Unfolding by Peptidylarginine Deiminase.J. Biol. Chem. 1996; 271: 30709-30716Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). PAD4 has been assigned more adverse functions, from regulation of gene-expression (7Cuthbert G.L. Daujat S. Snowden A.W. Erdjument-Bromage H. Hagiwara T. Yamada M. Schneider R. Gregory P.D. Tempst P. Bannister A.J. Kouzarides T. Histone deimination antagonizes arginine methylation.Cell. 2004; 118: 545-553Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 8Wang Y. Wysocka J. Sayegh J. Lee Y.-H. Perlin J.R. Leonelli L. Sonbuchner L.S. McDonald C.H. Cook R.G. Dou Y. Roeder R.G. Clarke S. Stallcup M.R. Allis C.D. Coonrod S.A. Human PAD4 regulates histone arginine methylation levels via demethylimination.Science. 2004; 306: 279-283Crossref PubMed Scopus (766) Google Scholar, 9Li P. Yao H. Zhang Z. Li M. Luo Y. Thompson P.R. Gilmour D.S. Wang Y. Regulation of p53 target gene expression by peptidylarginine deiminase 4.Mol. Cell. Biol. 2008; 28: 4745-4758Crossref PubMed Scopus (171) Google Scholar, 10Yao H. Li P. Venters B.J. Zheng S. Thompson P.R. Pugh B.F. Wang Y. Histone Arg modifications and p53 regulate the expression of OKL38, a mediator of apoptosis.J. Biol. Chem. 2008; 283: 20060-20068Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 11Zhang X. Gamble M.J. Stadler S. Cherrington B.D. Causey C.P. Thompson P.R. Roberson M.S. Kraus W.L. Coonrod S.A. Genome-wide analysis reveals PADI4 cooperates with Elk-1 to activate c-Fos expression in breast cancer cells.PLOS Genet. 2011; 7: 1-15Crossref Scopus (77) Google Scholar), through immune modulation (12Proost P. Loos T. Mortier A. Schutyser E. Gouwy M. Noppen S. Dillen C. Ronsse I. Conings R. Struyf S. Opdenakker G. Maudgal P.C. Van Damme J. Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation.J. Exp. Med. 2008; 205: 2085-2097Crossref PubMed Scopus (145) Google Scholar), to auto-citrullination for regulation of citrullinated protein (13Andrade F. Darrah E. Gucek M. Cole R.N. Rosen A. Zhu X. Autocitrullination of human peptidyl arginine deiminase type 4 regulates protein citrullination during cell activation.Arthritis Rheum. 2010; 62: 1630-1640Crossref PubMed Scopus (98) Google Scholar). Finally, the PAD6 function is not completely understood because of lack of substrate, but it is thought to have a role in reproduction (14Wright P.W. Bolling L.C. Calvert M.E. Sarmento O.F. Berkeley E.V. Shea M.C. Hao Z. Jayes F.C. Bush L.A. Shetty J. Shore A.N. Reddi P.P. Tung K.S. Samy E. Allietta M.M. Sherman N.E. Herr J.C. Coonrod S.A. ePAD, an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets.Dev. Biol. 2003; 256: 73-88Crossref PubMed Scopus (118) Google Scholar). humane peptidyl arginine deiminase outer membrane vesicles P. gingivalis W83 wild-type W83 knock-out mutant of peptidyl arginine deiminase W83 expressing PPAD with the active site cysteine mutated to alanine two-dimensional HFBA-based liquid chromatography mass spectrometry bacterial P. gingivalis peptidyl arginine deiminase rheumatoid arthritis prostaglandin E2 anti-citrullinated protein antibodies Arg-specific gingipains mass spectrometry heptafluorobutyric acid formic acid high performance liquid chromatography amino acid analysis Mascot Generic File format Mascot Generic eXtended retention time total ion count type IX secretion system. humane peptidyl arginine deiminase outer membrane vesicles P. gingivalis W83 wild-type W83 knock-out mutant of peptidyl arginine deiminase W83 expressing PPAD with the active site cysteine mutated to alanine two-dimensional HFBA-based liquid chromatography mass spectrometry bacterial P. gingivalis peptidyl arginine deiminase rheumatoid arthritis prostaglandin E2 anti-citrullinated protein antibodies Arg-specific gingipains mass spectrometry heptafluorobutyric acid formic acid high performance liquid chromatography amino acid analysis Mascot Generic File format Mascot Generic eXtended retention time total ion count type IX secretion system. Only few species of bacteria belonging to genus Porphyromonas have been found to express peptidyl arginine citrullinating enzyme closely related on the amino acid sequence level, if not identical. (15Gabarrini G. Chlebowicz M.A. Vega Quiroz M.E. Veloo A.C.M. Rossen J.W.A. Harmsen H.J.M. Laine M.L. van Dijl J.M. van Winkelhoff A.J. Conserved citrullinating exoenzymes in Porphyromonas species.J. Dent. Res. 2017; 97: 56-562Google Scholar) The best characterized is Porphyromonas gingivalis peptidyl arginine deiminase (PPAD). The sequence identity between PPAD and the human PADs is low, ∼30% (16Mangat P. Wegner N. Venables P.J. Potempa J. Bacterial and human peptidylarginine deiminases: targets for inhibiting the autoimmune response in rheumatoid arthritis?.Arthritis Res Ther. 2010; 12: 209Crossref PubMed Scopus (79) Google Scholar), however, similar activity is observed. There are some differences, however, and although human PADs activity is dependent on calcium and targets internal arginine residues, PPAD preferentially citrullinates C-terminal arginine in a calcium-independent manner (17Goulas T. Mizgalska D. Garcia-ferrer I. Kantyka T. Guevara T. Szmigielski B. Sroka A. Millán C. Usón I. Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase.Sci. Rep. 2015; 5: 1-17Crossref Scopus (59) Google Scholar). Apart from the preference for C-terminal arginine citrullination, specificity with respect to preceding residues has not been identified for PPAD. Conversely a study on PAD2 and PAD4 showed very broad specificity with PAD2 favoring Tyr in the +3 position (Assohou-Luy et al. (18Assohou-Luty C. Raijmakers R. Benckhuijsen W.E. Stammen-Vogelzangs J. De Ru A. Van Veelen P.A. Franken K.L.M.C. Drijfhout J.W. Pruijn G.J.M. The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities.Biochim. Biophys. Acta - Proteins Proteomics. 2014; 1844: 829-836Crossref PubMed Scopus (46) Google Scholar)). Citrullinations have been found to contribute to the pathogenicity of various diseases including rheumatoid arthritis (RA) and periodontitis. P. gingivalis, although absent or at the low level in dental biofilm of periodontally healthy subjects, occurs in high numbers in the mouth of periodontitis patients and is thought to be one of the primary causes of periodontitis (19Montgomery A.B. Kopec J. Shrestha L. Thezenas M.L. Burgess-Brown N.A. Fischer R. Yue W.W. Venables P.J. Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: Implications for autoimmunity in rheumatoid arthritis.Ann. Rheum. Dis. 2016; 75: 1255-1261Crossref PubMed Scopus (52) Google Scholar, 20Reichert S. Schlumberger W. Dähnrich C. Hornig N. Altermann W. Schaller H.G. Schulz S. Association of levels of antibodies against citrullinated cyclic peptides and citrullinated α - enolase in chronic and aggressive periodontitis as a risk factor of Rheumatoid arthritis : a case control study.J. Transl. Med. 2015; 18: 283Crossref Scopus (20) Google Scholar, 21Quirke A. Lugli E.B. Wegner N. Hamilton B.C. Charles P. Chowdhury M. Ytterberg A.J. Zubarev R.A. Potempa J. Culshaw S. Guo Y. Fisher B.A. Thiele G. Mikuls T.R. Venables P.J.W. Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase : a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis.Ann. Rheum. Dis. 2014; 73: 263-269Crossref PubMed Scopus (144) Google Scholar). Apart from PPAD P. gingivalis expresses Arg-specific gingipains, RgpA and RgpB (16Mangat P. Wegner N. Venables P.J. Potempa J. Bacterial and human peptidylarginine deiminases: targets for inhibiting the autoimmune response in rheumatoid arthritis?.Arthritis Res Ther. 2010; 12: 209Crossref PubMed Scopus (79) Google Scholar), which are important enzymes for citrullination, as they generate peptides and protein fragments with C-terminal Arg, instant substrates for modification by PPAD (22Wegner N. Wait R. Sroka A. Eick S. Nguyen K.-A. Lundberg K. Kinloch A. Culshaw S. Potempa J. Venables P.J. Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and alpha-enolase: implications for autoimmunity in rheumatoid arthritis.Arthritis Rheum. 2010; 62: 2662-2672Crossref PubMed Scopus (476) Google Scholar). In line with concerted action of Rgps and PPAD the Rgp-null mutant shows very little citrullination compared with the parental P. gingivalis W83 strain. Nevertheless, PPAD can modify internal arginine residues (23Abdullah S.N. Farmer E.A. Spargo L. Logan R. Gully N. Porphyromonas gingivalis peptidylarginine deiminase substrate specificity.Anaerobe. 2013; 23: 102-108Crossref PubMed Scopus (41) Google Scholar, 24Pyrc K. Milewska A. Kantyka T. Sroka A. Maresz K. Kozieł J. Nguyen K. Inactivation of epidermal growth factor by Porphyromonas gingivalis.Infect Immun. 2013; 81: 55-64Crossref PubMed Scopus (43) Google Scholar) but this reaction occurs at a rate thousand times slower than citrullination of C-terminal arginine (17Goulas T. Mizgalska D. Garcia-ferrer I. Kantyka T. Guevara T. Szmigielski B. Sroka A. Millán C. Usón I. Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase.Sci. Rep. 2015; 5: 1-17Crossref Scopus (59) Google Scholar). This is in accordance with the topology of the substrate binding site perfectly shaped to accommodate arginine at the C terminus with no room for an extended peptide chain (25Bereta G. Goulas T. Madej M. Bielecka E. Solà M. Potempa J. Gomis-rüth F.X. Structure, function, and inhibition of a genomic/clinical variant of Porphyromonas gingivalis peptidylarginine deiminase.Protein Sci. 2019; 28: 478-486Crossref PubMed Scopus (17) Google Scholar). Several findings implicate PPAD as an important virulence factor of P. gingivalis. Through citrullination of C-terminal residues in epidermal growth factor (24Pyrc K. Milewska A. Kantyka T. Sroka A. Maresz K. Kozieł J. Nguyen K. Inactivation of epidermal growth factor by Porphyromonas gingivalis.Infect Immun. 2013; 81: 55-64Crossref PubMed Scopus (43) Google Scholar) and C5a anaphylatoxin (26Bielecka E. Scavenius C. Kantyka T. Jusko M. Mizgalska D. Szmigielski B. Potempa B. Enghild J.J. Prossnitz E.R. Blom A.M. Potempa J. Peptidyl arginine deiminase from Porphyromonas gingivalis abolishes anaphylatoxin C5a activity.J. Biol. Chem. 2014; 289: 32481-32487Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) the enzyme can contribute to the periodontal tissue damage and attenuation of innate immune responses, respectively. Furthermore, P. gingivalis' citrullinome modulates neutrophil activity (27Kriebel K. Hieke C. Engelmann R. Potempa J. Muller-Hilke B. Lang H. Kreikemeyer B. Porphyromonas gingivalis peptidyl arginine deiminase can modulate neutrophil activity via infection of human dental stem cells.J. Innate Immun. 2018; 10: 264-278Crossref PubMed Scopus (8) Google Scholar), constrains P. gingivalis biofilm development (28Vermilyea D.M. Ottenberg G.K. Davey M.E. Citrullination mediated by PPAD constrains biofilm formation in P. gingivalis strain 381.NPJ biofilms microbiomes. 2019; 5: 7Crossref PubMed Scopus (15) Google Scholar), affects epithelial cells transcriptome (29Aliko A. Kaminska M. Bergum B. Gawron K. Benedyk M. Lamont R.J. Malicki S. Delaleu N. Potempa J. Mydel P. Impact of Porphyromonas gingivalis peptidylarginine deiminase on bacterial biofilm formation, epithelial cell invasion, and epithelial cell transcriptional landscape.Sci. Rep. 2018; 814144Crossref PubMed Scopus (18) Google Scholar), contributes to formation of dual-species biofilm with the opportunistic fungus Candidia (30Karkowska-Kuleta J. Bartnicka D. Zawrotniak M. Zielinska G. Kieronska A. Bochenska O. Ciaston I. Koziel J. Potempa J. Baster Z. Rajfur Z. Rapala-Kozik M. The activity of bacterial peptidylarginine deiminase is important during formation of dual-species biofilm by periodontal pathogen Porphyromonas gingivalis and opportunistic fungus Candida albicans.Pathog. Dis. 2018; 76Crossref PubMed Scopus (27) Google Scholar), and is responsible for stimulation of prostaglandin E2 (PGE2) secretion by gingival fibroblasts. (31Gawron K. Bereta G. Nowakowska Z. Lazarz-Bartyzel K. Lazarz M. Szmigielski B. Mizgalska D. Buda A. Koziel J. Oruba Z. Chomyszyn-Gajewska M. Potempa J. Peptidylarginine deiminase from Porphyromonas gingivalis contributes to infection of gingival fibroblasts and induction of prostaglandin E2 -signaling pathway.Mol. Oral Microbiol. 2014; 29: 321-332Crossref PubMed Scopus (22) Google Scholar) The latter activity can be directly linked to periodontitis and RA pathogenicity as PGE2 promotes bone resorption. Apart from that, C-terminally citrullinated peptides generated by concerted action of Rgp and PPAD are considered pivotal in breaking the immunotolerance leading to production of specific anti-citrullinated protein antibodies (ACPA) directly responsible for development of RA. (32Potempa J. Mydel P. Koziel J. The case for periodontitis in the pathogenesis of rheumatoid arthritis.Nat. Rev. Rheumatol. 2017; 13: 606-620Crossref PubMed Scopus (223) Google Scholar) This theory fits well, with the findings of heightened levels of ACPAs in periodontitis and RA patients. (20Reichert S. Schlumberger W. Dähnrich C. Hornig N. Altermann W. Schaller H.G. Schulz S. Association of levels of antibodies against citrullinated cyclic peptides and citrullinated α - enolase in chronic and aggressive periodontitis as a risk factor of Rheumatoid arthritis : a case control study.J. Transl. Med. 2015; 18: 283Crossref Scopus (20) Google Scholar, 21Quirke A. Lugli E.B. Wegner N. Hamilton B.C. Charles P. Chowdhury M. Ytterberg A.J. Zubarev R.A. Potempa J. Culshaw S. Guo Y. Fisher B.A. Thiele G. Mikuls T.R. Venables P.J.W. Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase : a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis.Ann. Rheum. Dis. 2014; 73: 263-269Crossref PubMed Scopus (144) Google Scholar, 33Lundberg K. Wegner N. Yucel-lindberg T. Venables P.J. Periodontitis in RA—the citrullinated enolase connection.Nat. Publ. Gr. 2010; 6: 727-730Crossref PubMed Scopus (257) Google Scholar, 34Laugisch O. Wong A. Sroka A. Kantyka T. Koziel J. Neuhaus K. Sculean A. Venables P.J. Potempa J. Möller B. Eick S. Citrullination in the periodontium - a possible link between periodontitis and rheumatoid arthritis.Clin. Oral Investig. 2016; 20: 675-683Crossref PubMed Scopus (65) Google Scholar, 35Haag S. Schneider N. Mason D.E. Tuncel J. Andersson I.E. Peters E.C. Burkhardt H. Holmdahl R. Identification of new citrulline-specific autoantibodies, which bind to human arthritic cartilage, by mass spectrometric analysis of citrullinated type ii collagen.Arthritis Rheumatol. 2014; 66: 1440-1449Crossref PubMed Scopus (47) Google Scholar, 36Pratesi F. Panza F. Paolini I. Petrelli F. Puxeddu I. Casigliani-Rabl S. Ancillotti D. Alcaro C. Rovero P. Migliorini P. Fingerprinting of anti-citrullinated protein antibodies (ACPA): Specificity, isotypes and subclasses.Lupus. 2015; 24: 433-441Crossref PubMed Scopus (11) Google Scholar) Likewise, it is supported by data from animal models of RA and P. gingivalis infection in which RA severity is dependent on PPAD expression. (32Potempa J. Mydel P. Koziel J. The case for periodontitis in the pathogenesis of rheumatoid arthritis.Nat. Rev. Rheumatol. 2017; 13: 606-620Crossref PubMed Scopus (223) Google Scholar) In this context, determination of P. gingivalis citrullinome is very important but a challenging task. The detection of citrullinations started with a color development reagent assay (COLDER assay) according to Clancy et al. (37Clancy K.W. Weerapana E. Thompson P.R. ScienceDirect Detection and identification of protein citrullination in complex biological systems.Curr. Opin. Chem. Biol. 2016; 30: 1-6Crossref PubMed Scopus (40) Google Scholar), which depends on chemical derivatization of the urea group of citrulline (38Knipp M. Vasák M. A Colorimetric 96-Well Microtiter Plate Assay for the Determination of Enzymatically Formed Citrulline.Anal. Biochem. 2000; 264: 257-264Crossref Scopus (165) Google Scholar). This method is mostly used in in vitro assays, because of its poor sensitivity and need of large amounts of citrullinations (37Clancy K.W. Weerapana E. Thompson P.R. ScienceDirect Detection and identification of protein citrullination in complex biological systems.Curr. Opin. Chem. Biol. 2016; 30: 1-6Crossref PubMed Scopus (40) Google Scholar). Antibody based methods also depend on chemical derivatization, the first anti-citrulline assay was developed by Senshu et al. (39Senshu T. Sato T. Inoue T. Akiyama K. Asaga H. Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane.Anal. Biochem. 1992; 203: 94-100Crossref PubMed Scopus (152) Google Scholar). These antibody-based assays have a major disadvantage as they have been found to cross-react with carbamylation, which is a chemical modification of lysine into homocitrulline (40Verheul M.K. van Veelen P.A. van Delft M.A.M. de Ru A. Janssen G.M.C. Rispens T. Toes R.E.M. Trouw L.A. Pitfalls in the detection of citrullination and carbamylation.Autoimmun. Rev. 2017; 17: 136-141Crossref PubMed Scopus (31) Google Scholar). Currently the most promising technique for the identification of citrullinations regarding sensitivity and specificity is mass spectrometry (MS). The development during the last decades within the MS field of protein research has made the investigation of clinical samples and whole-cell lysates possible, particularly when fractionated prior to injection. Further, less complex samples can be run directly, leading to significant reduction of manual work prior to analysis. The major problem with MS-based methods for the single Dalton mass shift is the possibility of misinterpretation of a deamination of asparagine or glutamine, picking a wrong isotope on the MS1 level, the loss of a single charge, giving rise to poor ionization and fragmentation, and small retention time shift. We addressed these problems in the present paper using heptafluorobutyric acid (HFBA) as the ion-pairing reagent during two-dimensional fractionation, as well as optimized mass spectrometric data acquisition, and development of specific software, Citrullia, designed for identification and validation of citrullinations. Cultures of P. gingivalis strain W83 and its isogenic mutants; C351A (with a point mutation of the catalytic cysteine residue, C351A, in PPAD rendering the enzyme catalytically inactive) and ΔPPAD (with the ppad gene deleted) were maintained on TSB agar plates with 5% defibrinated sheep blood and supplements: yeast extract (5 mg/ml), l-cysteine (0.5 mg/ml), hemin (5 μg/ml), and menadione (1 μg/ml). Liquid cultures were inoculated from 5 to 6 days old plates to liquid TSB medium with supplements and cultured for 18–20 h at 37 °C in an anaerobic chamber. Cultures were then diluted to OD600 = 0.1 in fresh medium and cultured as before for 20–22 h. Aldrithiol-4 (1.5 mm) was added to cultures immediately after incubation. Cultures were then centrifuged (7500 rcf, 15 min, 4 °C), supernatant collected and filtered through 0.45 μm membrane filter. The filtrate was ultracentrifuged at 70,000 rcf for 2 h at 4 °C. The collected sediment encompassing OMV was washed and then suspended in PBS with 1 mm TLCK by gentle sonication. The concentration of protein was determined using the Bradford method with bovine albumin as a standard. OMV were reduced by the addition of dithiothreitol (DTT) to a final concentration of 10 mm. The samples were then incubated for 30 min at 50–57 °C, followed by alkylation with iodoacetamide (IAA) added to a final concentration of 24 mm and incubation in the dark for 20 min. Excess IAA was removed by treatment with DTT and proteins in the sample were digested by overnight incubation at 37 °C with 2% w/w in-house methylated trypsin (41Heissel S. Frederiksen S.J. Id J.B. Peter H. Enhanced trypsin on a budget : Stabilization, purification and high-temperature application of inexpensive commercial trypsin for proteomics applications.PLoS One. 2019; 14e0218374Crossref PubMed Scopus (21) Google Scholar). For complete analysis without fractionation, the samples were micro purified essentially as described, (42Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 21896Crossref PubMed Scopus (2570) Google Scholar) dried down and resuspended in 0.1% formic acid (FA). For analysis by high performance liquid chromatography (HPLC) fractionation, each sample was dried down and resuspended in 0.05% heptafluorobutyric acid (HFBA) prior to off-line separation. Samples of 100 μg in a volume of 50 μl 0.05% HFBA were injected into an Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA) equipped with a Phenomenex Aeris™ 2.6 μm PEPTIDE XB-C18 100 Å, LC Column 150 × 2.1 mm (00F-4505-AN)(Phenomenex, Torrance, CA), running at a flow of 0.200 ml/min. Buffer A = 0.05% HFBA, buffer B = 0.05% HFBA and 90% acetonitrile. The method used a loading time of 2 min with 5% phase B and separated with a gradient of 12.5% B/5 min; 50.0% B/30 min, 60% B/35 min, 90%B/35.5 min, 90% B/36.5 min, 5% B/37 min, followed by equilibration at 5% B for 13 min. Samples were run on an EASY- nLC1000 Liquid Chromatography system (Thermo Fisher Scientific, Waltham, MA), using a 3 μm trap column (100 μm inner diameter, 5 μm Reprosilpur 120 C18, Dr. Maisch GmbH, Germany) and an 18 cm analytical column (75 μm inner diameter, 3 μm Reprosilpur 120 C18) coupled online to a Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific). The methods applied on the mass spectrometer had the following settings in common: positive mode, an MS1 resolution of 120,000, AGC target of 3e6, maximum injection time of 100 ms, and a scan range of 300–1400 m/z. The common MS2 settings were: resolution of 30,000, AGC target of 1e6, isolation window of 0.8 m/z, and a fixed first mass of 110.0 m/z. Furthermore, peptides with charges ranging from +1 to +6 were included, whereas +7 and above were excluded along with isotopes. For the duplicate experiment (30-min gradient) the following specific parameters were used: dynamic exclusion of 5 s, maximum injection