Large-Scale Protein Phosphorylation Analysis by Mass Spectrometry-Based Phosphoproteomics

Protein phosphorylation is one of the key mechanisms controlling cellular signaling networks. Due to the low abundance of phosphorylated proteins and weaker ionization efficiency of phosphopeptides during mass spectrometric analyses, it is highly required to remove abundant non-phosphopeptides from complex mixtures, such as cell lysates, allowing successful detection of low abundant phosphopeptides. We recently developed an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) to efficiently and selectively enrich phosphopeptides prior to mass spectrometry (MS) analysis. Here we describe a detailed workflow of HAMMOC for enriching phosphopeptides from small amounts, e.g., 100 μg, of tryptic digests of whole cell lysate. We also discuss the importance of confidently assigning phosphorylation site(s) from an identified phosphopeptide after MS analyses.