Fast, efficient, and quality-controlled phosphopeptide enrichment from minute sample amounts using titanium dioxide.

Bottom-up mass spectrometry (MS) is still the method of choice for analyzing protein phosphorylation. However, the low stoichiometry of phosphorylation, especially in highly complex samples, renders the specific enrichment of phosphopeptides prior to analysis inevitable. In recent years, specific phosphopeptide enrichment strategies combined with high-performance liquid chromatography (HPLC)-MS (LC-MS) provided researchers deeper insights into the phosphorylation networks of biological systems.Here, we describe two protocols for the enrichment of phosphopeptides from biological samples using titanium dioxide (TiO2) resins, enabling the handling of small sample amounts (<20 µg of protein) as well as larger sample amounts (up to the milligram range), depending on the scientific issue to be solved.Furthermore, we imply quality control steps during sample preparation to ensure the reproducibility and reliability of the phosphoproteomic findings.