title: Displacement of N/Q-rich peptides on TiO2 beads enhances the depth and coverage of yeast phosphoproteome analyses.
abstract: Phosphorylation is a reversible protein modification that regulates major cellular processes such as cell division, growth, and differentiation through highly dynamic and complex signaling pathways. Large-scale phosphoproteomics analyses have been greatly facilitated using affinity chromatography such as metal oxide affinity chromatography (e.g., TiO2), which in combination with mass spectrometry has enabled unbiased detection and quantification of thousands of phosphorylation sites in a single experiment. However, global phosphoproteome analyses do not provide comparable enrichment yields for different model organisms. While the proportion of phosphopeptides exceed 90% in mammalian cells using TiO2, similar levels have been notoriously difficult to achieve for yeast or dictylostelium cells. In a systematic study of TiO2 using cell extracts from different organisms, we determined that phosphopeptides are coenriched with peptides containing repetitive stretches of glutamine and asparagine residues. The proportion of these nonspecific binders can reach up to 50% in cell extracts from budding yeast and thus limit the depth and comprehensiveness of phosphoproteomics analyses. To address this limitation, we developed an effective method that used decoy amino acids to reduce the extent of nonspecific peptide binding and improve the recovery and detection of low abundance phosphopeptides that remained undetected by conventional TiO2 enrichment protocols.
authors.affiliation: Department of Biochemistry, Université de Montréal, Montréal, Quebec, Canada.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Kanshin
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Michnick
authors.forename: Pierre
authors.initials: P
authors.lastname: Thibault
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004331:Drosophila melanogaster / Q000737:chemistry; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D010750:Phosphoproteins / Q000302:isolation & purification*; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry*
chemical_list: D010455:Peptides; D010750:Phosphoproteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; C009495:titanium dioxide; C097188:polyglutamine; C045164:polyasparagine; D014025:Titanium