Chemical Approaches to Studying Labile Amino Acid Phosphorylation

Abstract

Phosphorylation of serine, threonine, and tyrosine residues is the archetypal posttranslational modification of proteins. While phosphorylation of these residues has become standard textbook knowledge, phosphorylation of other amino acid side chains is underappreciated and minimally characterized by comparison. This disparity is rooted in the relative instability of these chemically distinct amino acid side chain moieties, namely phosphoramidates, acyl phosphates, thiophosphates, and phosphoanhydrides. In the case of the O-phosphorylated amino acids, synthetic constructs were critical to assessing their stability and developing tools for their study. As the chemical biology community has become more aware of these alternative phosphorylation sites, methodology has been developed for the synthesis of well-characterized standards and close mimics of these phosphorylated amino acids as well. In this article, we review the synthetic chemistry that is a prerequisite to progress in this field.

Quest for the Crypto‐phosphoproteome

“…For studying the kinases and phosphatases, convenient and selective activity assays are essential. Although radiolabeling with [γ‐ 32 P]‐ATP has been widely used, it is not only hazardous and laborious, but it is difficult to distinguish the type of phosphorylation, requiring acid‐stability tests or phosphoamino acid analyses [8–10,16,17] . Recently developed pHis‐ or pArg‐specific antibodies enabled immunoblot‐based assays to circumvent these specificity issues [22,26–28,109] …”

“…Although radiolabeling with [γ-32 P]-ATP has been widely used, it is not only hazardous and laborious, but it is difficult to distinguish the type of phosphorylation, requiring acid-stability tests or phosphoamino acid analyses. [8][9][10]16,17] Recently developed pHis-or pArg-specific antibodies enabled immunoblot-based assays to circumvent these specificity issues. [22,[26][27][28]109] For convenient phosphatase assays, general substrates such as para-nitrophenyl phosphate (pNPP) and 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) [110] have been widely used.…”

“…Excellent reviews cover these advances, and we direct the readers to them for details. [14][15][16][17] In this Viewpoint, we will briefly survey the current status of the crypto-proteome research and discuss the missing pieces, concerning the phosphosites, kinases (writers), phosphatases (erasers), and phosphorylation-specific binding domains (readers), respectively ( Figure 2). [18]…”

Recent Updates on Protein N‐Phosphoramidate Hydrolases

“…Nevertheless, recent discoveries on labile PTMs and their hydrolases have been boosted by advances in chemical tools. For more comprehensive and in‐depth accounts of these tools, readers are directed to excellent recent reviews by the groups of Hackenberger and Fiedler …”

Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

“…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. Moderately 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).…”