Abstract
The regulatory role of protein cysteine phosphorylation is an under-researched area. The difficulty of accessing reference S-phosphorylated peptides (pCys-peptides) hampers progress in MS-driven cysteine phosphoproteomics, which requires targeted analytical procedures. This work describes an uncomplicated process for the conversion of disulfide-bridged protein into a complex model mixture of combinatorially modified peptides. Hen egg-white lysozyme was reduced with tris(2-carboxyethyl)phosphine (TCEP) followed by alkylation of cysteine with (3-acrylamidopropyl)trimethyl-ammonium chloride (APTA) and subsequent beta-elimination in aqueous Ba(OH)2 to yield modified polypeptides containing multiple dehydroalanine (Dha) residues. The conjugate addition of thiophosphoric acid to Dha residues followed by trypsinolysis led to numerous D/L phosphocysteine-containing peptides, which were identified by higher-energy collisional-dissociation tandem mass spectrometry (HCD-MS/MS). Our results show that some pCys-peptides produce prominent neutral losses of 80 Da, 98 Da and a weak 116 Da loss. These are similar to the neutral-loss triplets generated by phosphohistidine peptides.
Highlights
Continuing progress in phosphoproteomics studies is elucidating cellular processes that are modulated by dynamic phosphorylation on hydroxyl (Duan and Walther 2015), carboxyl (Attwood et al 2011; Lai et al 2017) and amine groups (Marmelstein et al 2017) in protein amino acid residues
Grade Modified Trypsin (Promega, V511A) the following reagents and solvents were purchased from Sigma-Aldrich and were used as received: 2-aminopyrazine (AP); 3-(acrylamidopropyl) trimethyl-ammonium chloride (APTA); acetonitrile (ACN); ammonium bicarbonate; barium carbonate; barium hydroxide; 2,5-dihydroxybenzoic acid (DHB); formic acid (FA); guanidine hydrochloride (GdnâHCl); HCl; lysozyme from hen egg-white; N-methylmaleimide; trans-3,5-dimethoxy4-hydroxycinnamic acidâsinapinic acid (SA); solid carbon dioxide, trifluoroacetic acid (TFA); Tris; tris-carboxymethyl phosphine (TCEP); urea
It was assumed that polypeptides bearing cysteine residue S-protected by other simple alkyl groups might produce ÎČ-elimination products in amounts detectable by modern mass spectrometry
Summary
Continuing progress in phosphoproteomics studies is elucidating cellular processes that are modulated by dynamic phosphorylation on hydroxyl (Duan and Walther 2015), carboxyl (Attwood et al 2011; Lai et al 2017) and amine groups (Marmelstein et al 2017) in protein amino acid residues. Susceptible to acid hydrolysis, S-linked phosphorylation is among the least explored modifications (Shannon and Weerapana 2013; Piggott and Attwood 2017) and it is discussed mainly in the context of transient cysteine phosphorylation of protein tyrosine phosphatases (Tonks 2014). In 2012, the reversible cysteine phosphorylation of Handling Editor: J.
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