Specific biotinylation and sensitive enrichment of citrullinated peptides
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Protein citrullination is a posttranslational modification where peptidylarginine is enzymatically deiminated to form peptidylcitrulline. Although the role of protein citrullination in both health and disease is being increasingly recognised, techniques available to identify citrullinated proteins and to map their citrullination site(s) are rare and often show poor sensitivity. Here, we present a sensitive technique for specific modification and selective enrichment of citrullinated peptides from complex biological samples. The technique is based on highly specific in-solution biotinylation of citrulline residues followed by selective enrichment of modified peptides using streptavidin beads. We demonstrate that a synthetic citrulline-containing peptide can be selectively enriched when less than 0.5 pmol is spiked into a highly heterogeneous peptide mixture. After enrichment, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of an aliquot of the streptavidin eluate corresponding to theoretically 50 fmol of the spiked-in peptide showed a prominent signal. We further demonstrate the sensitivity of our technique by enrichment of citrullinated peptides from enzymatically deiminated myelin basic protein (MBP), when 10 pmol was spiked into a heterogeneous biological digest. In MALDI-TOF MS analysis, six MBP-derived citrullinated peptides were observed, showing the efficiency of this enrichment strategy. The high sensitivity combined with the remarkable specificity of the described technique makes it a valuable tool for elucidating citrullination in various biological processes.
KeywordsProtein citrullination Chemical modification Biotinylation Peptide enrichment Rheumatoid arthritis Mass spectrometry
We thank Fridtjof Lund-Johansen for providing the A431 cell line and Maria Stensland for providing recombinant human PAD4.We further thank Dr. Gustavo de Souza for critical reading of the manuscript and discussions. Samples were analysed by A.E.V.T. at the Proteomics Core Facility, OUS-UiO, which is supported by an Infrastructure grant from the South-East Health Authority. This research was supported by grants from the MLSUiO-Molecular Life Science, University of Oslo to A.E.V.T. and from the Research Council of Norway to A.H. and B.F.
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