PDF《peptides†》

On-line liquid chromatography neutral loss-triggered electron transfer dissociation mass spectrometry for the targeted analysis of citrullinated peptides? Andrew J.

Creese,ab Melissa M. Grant,a Iain L. C. Chapplea and Helen J. Cooper*b Received 29th June 2010, Accepted 5th November

2010 DOI: 10.1039/c0ay00414f Citrullination is a post-translational modi?cation of proteins which deiminates arginine, increasing the mass by 0.98 Da. Protein citrullination is a known biomarker for multiple sclerosis and a potential biomarker for rheumatoid arthritis. Collision-induced dissociation (CID) tandem mass spectrometry of citrullinated peptides produces a dominant neutral loss of isocyanic acid (HNCO, ?43 Da) from the deiminated arginine amino acid side-chain. Here we show that the loss of isocyanic acid in CID can be used as a trigger for targeted analysis by supplemental activation electron transfer dissociation (saETD). Unlike CID, post-translational modi?cations (PTMs) are retained on peptide backbone fragments produced by saETD, improving the con?dence in assignment of both peptide sequence and PTM site. The method is demonstrated for four synthetic peptides spiked into complex trypsin-digested saliva samples and a commercial six protein tryptic mixture. In contrast to CID alone, the neutral-loss triggered ETD approach results in high con?dence identi?cation of three of the four peptides, including an unexpected disul?de-bound dimer, and zero false positives. Introduction Citrullination is a post-translational modi?cation (PTM) of proteins in which an arginine amino acid residue is converted to citrulline via deimination, see Scheme 1. The modi?cation has the same mass increase (+0.9840 Da) as deamidation of asparagine or glutamine residues.1 The citrullination of arginine is caused by a group of calcium dependent enzymes called peptidylarginine deiminases (PADs).2 The conversion of arginine to citrulline decreases the isoelectric point (pI) of the protein and affects its structure and function. Increased levels of citrullinated myelin basic protein have been detected in the brains of patients suffering from multiple sclerosis3,4 and Alzheimer'

s disease.5 Citrulline-modi?ed proteins vimentin, ?bronectin and a-enolase have also been detected as part of the in?ammatory-immune response in rheumatoid arthritis (RA).6C8 The modi?cation of these proteins is used as a serum biomarker for RA.6 In addition to those described above, the histones9,10 H3, H2A and H4 and myosin11 have been shown to be citrullinated. The apparent speci?city of citrullination increases its potential as a disease biomarker. A signi?cant association has been demonstrated between periodontitis and rheumatoid arthritis in epidemiological research,12 but speci?c mechanistic links between these two chronic in?ammatory diseases have not been fully elucidated.12 One of the principal pathogenic bacteria in periodontitis, Por- phyromonas gingivalis,13 is thought to also play a role in RA.14 P. gingivalis is the only known periodontal bacterium to contain a PAD enzyme15 and this organism is found in both the gingival crevicular ?uid (GCF)16 and saliva17 of patients with perio- dontitis. Saliva may therefore be a suitable and convenient substrate for the non-invasive diagnosis of RA. Tandem mass spectrometry-based proteomics methods are routinely used to identify the peptides and proteins present in a biological sample18C20 and to characterise any post-trans- lational modi?cations.21,22 A standard proteomics approach is to digest a protein mixture with trypsin and use on-line reversed- phase liquid chromatography (LC) to separate the peptides.23,24 The LC is coupled to collision induced dissociation (CID)25 tandem mass spectrometry (MS/MS). CID fragments peptide ions by cleaving the NCC0 bond producing b and y26 fragment ions, thus providing sequence information which can be searched against protein databases. Algorithms are employed to match the measured peptide mass spectrum with those calculated from in silico digests of proteins contained in a database. The peptide assignment is scored according to the similarity between the experimental data and the theoretical mass spectrum. Using this approach, it is possible to identify thousands of peptides (and proteins) from an LC-MS/MS analysis.27 CID fragmentation is a thermal process: one of the drawbacks of CID is the Scheme