Chip-Based Enrichment and NanoLC−MS/MS Analysis of Phosphopeptides from Whole Lysates

Protein phosphorylation may be the most widespread and possibly most important post-translational modification (PTM). Considering such a claim, it should be no surprise that huge efforts have been made to improve methods to allow comprehensive study of cellular phosphorylation events. Nevertheless, comprehensive identification of sites of protein phosphorylation is still a challenge, best left to experienced proteomics experts. Recent advances in HPLC chip manufacturing have created an environment to allow automation of popular techniques in the bioanalytical world. One such tool that would benefit from the increased ease and confidence brought by automated ‘nanoflow’ analysis is phosphopeptide enrichment. To this end, we have developed a reusable HPLC nanoflow rate chip using TiO₂ particles for selective phosphopeptide enrichment. Such a design proved robust, easy to use, and was capable of consistent performance over tens of analyses including minute amounts of complex cellular lysates.