Optimizing the Identification of Citrullinated Peptides by Mass Spectrometry: Utilizing the Inability of Trypsin to Cleave after Citrullinated Amino Acids

Bennike, Tue Bjerg; Lauridsen, Kasper; Olesen, Michael Kruse; Andersen, Vibeke; Birkelund, Svend; Stensballe, Allan

doi:10.4172/jpb.1000293

Cited by 36 publications

(58 citation statements)

References 27 publications

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“…The results of the peptide microarray were verified and extended by ELISA for the RA candidate autoantigens Cit-Vim 60-75 , Table 1. Characteristics of the human anti-citrullinated protein mAb generated from synovial fluid plasma cells from a rheumatoid arthritis patient* Cit-Fibβ 36-52 , Cit-Fil 307-324 , Cit-H4 , Cit-H4 31-50 , Cit-H3 1-30 , and CEP-1 [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] , demonstrating that some of the citrulline peptides were detected at an mAb concentration as low as 5-10 ng/ml ( Figure 1B and Supplementary Figure 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/ doi/10.1002/art.40699/abstract). The equilibrium constants (KDs) for binding to selected citrulline peptides were determined by SPR assay (of note, mAb 1325:07E07 was not analyzed by SPR assay because of the limited amounts obtained in our expression system).…”

Section: Resultsmentioning

confidence: 99%

“…Lack of binding was defined as a sensogram showing such low affinity that no equilibrium constant could be determined. NA = not analyzed; CDR3 = third complementaritydetermining region; Cit-Fil 307-324 = citrullinated filaggrin 307-324 ; Cit-Fibβ 36-52 = citrullinated fibrinogen β 36-52 ; NB = no binding; Cit-Vim 60-75 = citrullinated vimentin 60-75 ; CEP-1 5-21 = citrullinated α-enolase peptide[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] ; Arg-Fibβ 36-52 = arginine-containing fibrinogen β 36-52 ; Arg-Vim 60-75 = arginine-containing vimentin 60-75 ; REP-1 5-21 = arginine-containing α-enolase peptide[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] .…”

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confidence: 99%

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Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell–Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis

Stéen

Forsström

Sahlström

et al. 2019

Arthritis & Rheumatology

105

169

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Our findings suggest that the high level of cross reactivity amongst the RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is in line with the notion that RA is initiated in one context, such as in mucosal organs, and thereafter target other sites, such as joints. This article is protected by copyright. All rights reserved.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell–Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis

Stéen

Forsström

Sahlström

et al. 2019

Arthritis & Rheumatology

105

169

View full text Add to dashboard Cite

show abstract

“…Profiling the substrate specificity of PAD isoforms will generate insight for developing selective inhibitors . Previous assays have used trypsin to detect citrullination with mass spectrometry because it cleaves after arginine residues but not citrulline residues . However, trypsin also cleaves lysine residues, which complicates the analysis of substrates containing a mixture of trypsin cleavage sites.…”

Section: Discussionmentioning

confidence: 99%

An Immobilized Enzyme Reactor for Spatiotemporal Control over Reaction Products

Grant

Modica

Roll

et al. 2018

Small

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This paper describes a microfluidic chip wherein the position and order of two immobilized enzymes affects the type and quantity of reaction products in the flowing fluid. Assembly of the chip is based on a self-assembled monolayer presenting two orthogonal covalent capture ligands that immobilize their respective fusion enzyme. A thiol-tagged substrate is flowed over a region presenting the first enzyme-which generates a product that is efficiently transferred to the second enzyme-and the second enzyme's product binds to an adjacent thiol capture site on the chip. The amount of the three possible reaction products is quantified directly on the chip using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry, revealing that the same microsystem can be spatiotemporally arranged to produce different products depending on the device design. This work allows for optimizing multistep biochemical transformations in favor of a desired product using a facile reaction and analytical format.

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“…The citrullination of arginine makes the residue heavier by 0.984 Dalton and become resistant to trypsin digestion [23, 24]. Thus, we considered cleavable arginine to be un-citrullinated.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Autoantigens Targeted by Anti-Citrullinated Protein Antibodies In Vivo: Prominent Role for Epitopes Derived from Histone 4 Proteins

Meng¹,

Ezzati²,

Smolik³

et al. 2016

PLoS ONE

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Anti-citrullinated protein antibodies (ACPA) have become an integral part of the clinical definition of rheumatoid arthritis, and are hypothesized to be important in the immunopathogenesis of this autoimmune disease. Several citrullinated proteins have been demonstrated to serve as candidate autoantigens for the ACPA, based on in vitro immune reactions between citrullinated peptides/proteins and RA sera. Yet it remains unclear whether the autoantigens identified in vitro are indeed directly and specifically targeted by the ACPA in vivo. Moreover, it is unclear whether ACPA present in RA sera are directed towards the same spectrum of autoantigens as the ACPA present within the synovial compartment. In this study, we isolated ACPA immune complexes from RA synovial fluids (SF) and sera by using immobilized cyclic citrullinated peptides (CCP3) based immune affinity, and characterized the proteins that are directly and specifically associated with them by mass spectrometry. The results demonstrate that four histone proteins are prominent ACPA autoantigens, with the frequency of detection being histone H4 (89%), H2B (63%), H3 (63%), and H2A (58%) in ACPA positive RA SF. We further demonstrate that a histone 4 peptide containing citrulline at position Cit39 was recognized by 100% of ACPA positive RA SF. An adjacent citrulline residue at Cit40 was recognized by 34% of ACPA positive RA SF. An H4 peptide containing Cit39-40 was recognized in the serum of 94% ACPA positive RA, 77% ACPA positive first-degree relatives (FDR) of RA patients, and 2.5% of healthy controls. The Cit39-40 peptide substantially blocked the ACPA reactivity in both SF and serum. Although the spectrum of ACPA we identified was limited to those isolated using immobilized CCP3 peptides, the findings indicate that H4 is a widely recognized RA autoantigen in both the synovial and serum compartments. The identification of this immunodominant ACPA epitope may be valuable in designing approaches to immune tolerance induction in RA.

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Optimizing the Identification of Citrullinated Peptides by Mass Spectrometry: Utilizing the Inability of Trypsin to Cleave after Citrullinated Amino Acids

Cited by 36 publications

References 27 publications

Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell–Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis

Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell–Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis

An Immobilized Enzyme Reactor for Spatiotemporal Control over Reaction Products

Characterization of Autoantigens Targeted by Anti-Citrullinated Protein Antibodies In Vivo: Prominent Role for Epitopes Derived from Histone 4 Proteins

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