P65-M Phosphopeptide Enrichment Using a High-Affinity Metal Oxide Sorbent Packed in a Micro-Elution Plate

摘要

The reversible phophorylation of serine, threonine, and tyrosine is one of the most important post-translational modifications involved in various cellular functions. Identification of phophorylation sites by mass spectrometry is challenging due to the low abundance of phosphopeptides and their limited ionization efficiency. Therefore, it is critical to selectively enrich the phosphopeptides prior to MS analysis. In this study we investigated the affinity extraction of phosphopeptides using a metal oxide–based solid-phase extraction (SPE) microscale device. This sorbent shows high affinity towards phosphopeptides; acidic peptide adsorption is greatly minimized. When dealing with highly complex samples (e.g., cell lysate), the selectivity of phosphopeptides can be further improved by mixing the sample with additive, aromatic carboxylic acids prior to loading onto the SPE. The metal oxide SPE is compared with other methods such as immobilized metal affinity chromatography and titanium dioxide–based phosphopeptide enrichment. Enhanced performance was observed in terms of the selectivity. MALDI-TOF and nanoLC/MS/ MS were used to study the phosphopeptide recovery and selectivity. Phosphopeptide standards, alpha-casein tryptic digest, and yeast cell lysate were used to evaluate the phosphopeptide enrichment method’s performance.