Targeted analysis of protein citrullination using chemical modification and tandem mass spectrometry.
Protein citrullination originates from enzymatic deimination of polypeptide-bound arginine and is involved in various biological processes during health and disease. However, tools required for a detailed and targeted proteomic analysis of citrullinated proteins in situ, including their citrullination sites, are limited. A widely used technique for detection of citrullinated proteins relies on antibody staining after specific derivatization of citrulline residues by 2,3-butanedione and antipyrine. We have recently reported on the details of this reaction. Here, we show that this chemical modification can be utilized to specifically detect and identify citrullinated peptides and their citrullination sites by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Using model compounds, we demonstrate that in collision-induced dissociation (CID) a specific, modification-derived fragment ion appears as the dominating signal at m/z 201.1 in the MS/MS spectra. When applying electron transfer dissociation (ETD), however, the chemical modification of citrulline remained intact and extensive sequence coverage allowed identification of peptides and their citrullination sites. Therefore, LC/MS/MS analysis with alternating CID and ETD has been performed, using CID for specific, signature ion-based detection of derivatized citrullinated peptides and ETD for sequence determination. The usefulness of this targeted analysis was demonstrated by identifying citrullination sites in myelin basic protein deiminated in vitro. Combining antibody-based enrichment of chemically modified citrulline-containing peptides with specific mass spectrometric detection will increase the potential of such a targeted analysis of protein citrullination in the future.
Stensland
Maria
M
Centre for Immune Regulation, Institute of Immunology, University of Oslo, Rikshospitalet University Hospital, 0027 Oslo, Norway.
Holm
Anders
A
Kiehne
Andrea
A
Fleckenstein
Burkhard
B
D000818:Animals; D002417:Cattle; D002956:Citrulline / Q000737:chemistry*; D006801:Humans; D006867:Hydrolases / Q000737:chemistry*; D004676:Myelin Basic Protein / Q000737:chemistry*; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D012709:Serum Albumin / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004676:Myelin Basic Protein; D012709:Serum Albumin; D002956:Citrulline; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases