Tandem Mass Tag Approach Utilizing Pervanadate BOOST Channels Delivers Deeper Quantitative Characterization of the Tyrosine Phosphoproteome

There are clear advantages to experimental approaches that reduce the amount of necessary cellular material for wide-scale quantitative profiling of the tyrosine phosphoproteome, particularly in biological systems where material is limited. The incorporation of boost channels in a multiplexed tandem mass tag experiment containing cells treated with the broad-spectrum tyrosine phosphatase inhibitor pervanadate enabled up to 6.3-fold deeper quantification of the tyrosine phosphoproteome while maintaining accuracy and precision. [Display omitted] Highlights •Detection of low-abundance phosphotyrosine-containing peptides is challenging.•Multiplexed TMT allows inclusion of modification(pTyr)-saturated boost channels.•Boost channels facilitate selection of pTyr precursor ions for fragmentation.•Quantitation depth is increased while maintaining accuracy and precision. Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers' ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, the BOOST (Broad-spectrum Optimization Of Selective Triggering) method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of pervanadate (PV) boost channels (cells treated with the broad-spectrum tyrosine phosphatase inhibitor PV) selectively increased the relative abundance of pTyr-containing peptides. After PV boost channels facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered accurate quantitation of each peptide for the experimental samples while the quantitation from PV boost channels was ignored. This method yielded up to 6.3-fold boost in pTyr quantification depth of statistically significant dat