Phosphoproteomics—More than meets the eye

Abstract

PTMs enable cells to adapt to internal and external stimuli in the milliseconds to seconds time regime. Protein phosphorylation is probably the most important of these modifications as it affects protein structure and interactions, critically influencing the life cycle of a cell. In the last 15 years, new insights into phosphorylation have been provided by highly sensitive MS‐based approaches combined with specific phosphopeptide enrichment strategies. Although so far research has mainly focused on the discovery and characterization of O‐phosphorylation, this review also briefly outlines the current knowledge about N‐phosphorylation depicting its ubiquitous relevance. Further, common pitfalls in sample preparation, LC‐MS analysis, and subsequent data analysis are discussed as well as issues regarding quality and comparability of studies on protein phosphorylation.

Signal Transduction: From the Atomic Age to the Post-Genomic Era

“…The protein–protein interactions dependent on such modifications are generally dynamic because these groups are installed and removed by enzymes whose own activity often depends on signaling—in particular, protein phosphorylation (Hunter 2012; Jin and Pawson 2012)—which makes the study of protein kinases and phosphoprotein phosphatases central to our understanding of signal transduction (Cohen 2002; Fischer 2013). This area of research has driven development of new tools for globally interrogating both the kinome (Knight et al 2013) and the phosphoproteome (Leitner et al 2011), including position-oriented, combinatorial, synthetic peptide libraries (Turk et al 2006; Arsenault et al 2011) and immobilized whole proteome arrays (“protein chips”) (Ptacek and Snyder 2006) for delineating kinase-substrate specificity, ever more specific small-molecule kinase inhibitors (Cohen and Alessi 2013), genetic approaches to uniquely sensitize a given kinase to inhibition (Elphick et al 2007; Knight and Shokat 2007; Feldman and Shokat 2010; Kliegman et al 2013), selective chemical tags to covalently label particular kinases and phosphatases or their substrates (Allen et al 2007; Patricelli et al 2007; Hertz et al 2010; Sadowsky et al 2011; Miller et al 2013), yeast two-hybrid screens (Cook et al 1996; Fukada and Noda 2007; Sopko and Andrews 2008), and other strategies to trap particular kinases and phosphatases in complexes with their targets (Blanchetot et al 2005; Boubekeur et al 2011), as well as sophisticated mass spectrometry instrumentation and corresponding methods for detecting and cataloging phosphoproteins (Cohen and Knebel 2006; Chi et al 2007; Gevaert and Vandekerckhove 2009; Palumbo et al 2011; Engholm-Keller and Larsen 2013; Loroch et al 2013; Roux and Thibault 2013). …”

Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveal chaperone-dependent regulation of ribonucleotide reductase

“…It is thus easier to examine enriched or fractionated proteomes. This includes PTM enrichment such as phosphoprotein or phosphopeptide purification [27, 28]. Fractionation by cellular compartment is another way to simplify the proteome [29, 30].…”

Understanding cell signaling in cancer stem cells for targeted therapy – can phosphoproteomics help to reveal the secrets?

“…Furthermore, it is important to localize the phosphorylation to the corresponding amino acid residue. This phosphosite localization can be even more important and challenging than the peptide identification itself craving for an appropriate algorithm [87]. …”