Enrichment of Phosphorylated Peptides and Proteins by Selective Precipitation Methods

Abstract

Protein phosphorylation is one of the most prominent post-translational modifications involved in the regulation of cellular processes. Fundamental understanding of biological processes requires appropriate bioanalytical methods for selectively enriching phosphorylated peptides and proteins. Most of the commonly applied enrichment approaches include chromatographic materials including Fe(3+)-immobilized metal-ion affinity chromatography or metal oxides. In the last years, the introduction of several non-chromatographic isolation technologies has increasingly attracted the interest of many scientists. Such approaches are based on the selective precipitation of phosphorylated peptides and proteins by applying various metal cations. The excellent performance of precipitation-based enrichment methods can be explained by the absence of any stationary phase, resin or sorbent, which usually leads to unspecific binding. This review provides an overview of recently published methods for the selective precipitation of phosphorylated peptides and proteins.

Carbon nanostructure morphology templates nanocomposites for phosphoproteomics

“…Addition and removal of phosphate groups offers the means to regulate cellular functions, and it is altered in disease processes [13]. Unfortunately, phosphorylated forms of biomolecules are transient, thus their characterization constitutes a key analytical challenge in shotgun proteomics analysis, especially of complex samples [14][15][16]. Amongst the different strategies that have been devised to address this issue, effective approaches consist of immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) [17].…”

Development of magnetic ytterbium oxide core–shell particles for selectively trapping phosphopeptides

“…Particularly lanthanide metal ions and lanthanide oxides have successfully been applied in phosphoproteomics. 47,48 This can be explained by the strong preference of lanthanides for oxygen-containing anions like phosphates, with which they form very stable phosphoprotein complexes. 49 The immobilization of phosphopeptides is accomplished through bidentate ligand coordination.…”

Recent findings and technological advances in phosphoproteomics for cells and tissues

“…A number of alternative enrichment strategies have been developed in recent years, including polymer-based metal ion affinity capture (PolyMAC) [43,44] , Phos-tag [45] , as well as precipitation-based enrichment strategies, such as those based on lanthanide ions [46] . Those techniques are further complemented with targeted strategies enriching specific subsets of the phosphoproteome, such as motif-specific antibodies [47] , or ‘fishing’ with phospho-binding domains such as phosphotyrosine binding SH2 domains [48] or 14–3–3 motifs [49] .…”