Journal
JOURNAL OF PROTEOMICS
Volume 91, Issue -, Pages 331-337Publisher
ELSEVIER
DOI: 10.1016/j.jprot.2013.07.024
Keywords
Tyrosine phosphorylation; Immuno-affinity enrichment; Ti4+-IMAC; Phosphoproteomics; K562 cells; Hela cells
Categories
Funding
- PRIME-XS project [262067]
- European Union
- Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative
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Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteornics remains challenging, due to the low occurrence of this post-translational modification compared to serine and threonine phosphorylation events in mammalian systems. Conventional metal-based affinity chromatography methods used to enrich phosphopeptides can nowadays isolate over 10,000 phosphopeptides. However, these approaches are not particularly suitable for the selective enrichment of low abundant Tyr phosphorylated peptides as the higher abundant co-enriched serine (Ser) and threonine (Thr) phosphorylated peptides typically obscure their detection. Therefore, a more targeted approach based on immuno-affinity precipitation at the peptide level has been introduced for the specific analysis of Tyr phosphorylated species. This method typically leads to the detection of a few hundreds of phosphopeptides, albeit typically over 70% of those are Tyr phosphorylated. Here, we evaluated and compared phosphotyrosine peptides enriched by a phospho-Tyr immuno-affinity enrichment (employing pY99 antibodies) and a multidimensional approach consisting of metal affinity based enrichment (Ti4+-IMAC) followed by hydrophilic interaction liquid chromatography (HILIC) fractionation. Our aim was to assess differences and similarities in the set of Tyr phosphorylated peptides detected by each approach. Our data suggest that both strategies are not redundant but complementary and should ideally be combined for a more comprehensive view at phosphotyrosine signaling. Biological significance Here we evaluated enabling tools for the global analysis of phosphotyrosine phosphorylation. Phosphotyrosine phosphorylation is a key protein modification driving cellular response also involved in disease/cancer molecular pathways. (C) 2013 Elsevier B.V. All rights reserved.
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