Over the past decade phosphoproteomics has become an emerging discipline within proteomics research, focusing on detection of the reversible modification of proteins by phosphorylation of serine, threonine, and tyrosine residues. For successful analysis, phosphopeptide enrichment is often a prerequisite due to their low stoichiometry, heterogeneity, and low abundance. The enrichment of phosphopeptides is often performed manually, which is inherently labor intensive and a major hindrance in large-scale analyses. Automation of the enrichment method would vastly improve reproducibility and thereby facilitate “high-throughput” phosphoproteomics research. Here, we describe the setup of a simple, robust, and automated online TiO2-based nanoscale chromatographic approach to selectively enrich and separate phosphorylated peptides from proteolytic digests of moderate and high complexity.
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The authors would like to acknowledge GL Sciences Inc., Tokyo, for the TiO2 Titansphere material. This work was supported by the Netherlands Proteomics Centre (http://www.netherlandsproteomicscentre.nl), a program embedded in the Netherlands Genomics Initiative.
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