Puzzling over protein cysteine phosphorylation – assessment of proteomic tools for S-phosphorylation profiling

Abstract

Cysteine phosphorylation has recently been discovered in both prokaryotic and eukaryotic systems, and is thought to play crucial roles in signaling and regulation of cellular responses. This article explores the topics of chemical stability of this type of structural modification and the resulting issues regarding affinity enrichment of S-phosphopeptides and their mass spectrometry-based detection in the course of general proteomics studies. Together, this work suggests that the current advances in phosphoproteomic methodologies provide adequate tools for investigating protein cysteine phosphorylation and appear to be immediately available for practical implementation. The article provides useful information necessary for designing experiments in the emerging cysteine phosphoproteomics. The examples of methodological proposals for S-linked phosphorylation detection are included herein in order to stimulate development of new approaches by the phosphoproteomic community.

Protein phosphorylation and its role in archaeal signal transduction

“…2014) or as part of the enzyme mechanism in protein-tyrosine phosphatases (Fuhrmann et al. 2009; Buchowiecka 2014). Phosphorylation of the positive charged amino acid Arg and Lys forms a high-energy phosphoramidate bond.…”

Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides

“…Extensive studies of phosphorylation, which mainly occurs on serine, threonine and tyrosine amino-acid side chains, have revealed important insights into protein functions. The phosphorylation of other amino acids, such as phospho-histidine (pHis), -arginine (pArg), -lysine (pLys) and -cysteine (pCys), is less understood and studies of these events have been hindered by technical limitations 3 4 5 6 7 . The main obstacle to studying N - and S -phosphorylation has been their acid-lability, which prevents their accessibility and characterization through standard chemical and analytical tools.…”

“…These chemical tools have permitted the development of mass spectrometry (MS)-based methods or the generation of antibodies, leading to a better understanding of the biological role of these modifications 14 15 16 . The isolation and characterization of phosphorylated-Cys peptides, however, has been challenging due to the acid-lability of the phosphorothiolate bond, which has prevented further identification of unknown pCys sites using standard phosphoproteomic approaches 7 .…”

Phosphorus-containing amino acids with a P–C bond in the side chain or a P–O, P–S or P–N bond: from synthesis to applications

“…It is of major importance to understand the role of S-phosphocysteine in the regulation of tyrosine phosphorylation, which is associated with cell growth and division processes and is therefore of interest to cancer researchers. [106][107][108] Only a few methodologies have been developed for the synthesis of S-phosphocysteine derivatives. The rst strategy is based on a Michaelis-Arbuzov reaction involving a readily available disulfanyl derivative and trimethyl phosphite, leading to the corresponding phosphorothioate 356 with an almost quantitative yield aer purication (Scheme 81).…”