Analytical strategies for phosphoproteomics

Abstract

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

Global Analysis of Muscle-specific Kinase Signaling by Quantitative Phosphoproteomics

“…However, proteins such as docking protein-7, CT10-regulated kinase or Src (40,41) that are known to be tyrosine phosphorylated downstream of MuSK were missing from our dataset. Using an IMAC approach to characterize global phosphorylation events favors the enrichment of phosphoserine and phosphothreonine peptides because MS analysis of complex samples is biased toward more abundant peptides and is therefore likely to miss phosphotyrosine peptides, which occur at low abundance (42)(43)(44). This challenge can be overcome by immunoprecipitations using antibodies against phosphotyrosine to specifically enrich tyrosine phosphorylated peptides or proteins.…”

Perspectives of Comprehensive Phosphoproteome Analysis Using Shotgun Strategy

“…Chemical derivatization strategies, such as β-elimination coupled with Michael addition and phosphoramidate chemistry (PAC), can also be applied for phosphopeptides enrichment, but the performance is usually inferior to IMAC and metal oxide. 9 Recently, nanosize polymer based metal ion affinity capture (PolyMAC) was developed, and high reproducibility, selectivity, and recovery for phosphopeptide enrichment were obtained. 21,22 Phosphotyrosine dependent networks play a key role in transmitting signals, which is important for elucidating the regulatory mechanisms of each biological effect.…”

Role of proteomics in nutrigenomics and nutrigenetics

“…Current mass spectrometry (MS)-based proteomic platforms can deliver a dynamic range of 10 4 . This means that the remaining, as such inaccessible, low-abundance proteome has to be addressed by either depletion of the most-abundant proteins (e.g., by the commercially available multiple affinity removal system that specifically removes the top seven or even 14 plasma proteins) [17], or by selective enrichment of low-abundance proteins (e.g., by the immobilized metal affinity chromatography or titanium dioxide techniques for phosphoproteins [18], lectins [19] or the cell-surface capture technique for glycoproteins [20]). Another way of dealing with the dynamic range is 'focus with a gain in depth at the expense of breadth', and this is based on targeted MS analysis as discussed in the following sections.…”