Characterization of Biases in Phosphopeptide Enrichment by Ti4+-mobilized Metal Affinity Chromatography and TiO2 Using a Massive Synthetic Library and Human Cell Digests

Outcomes of comparative evaluations of enrichment methods for phosphopeptides depend highly on the experimental protocols used, the operator, the source of the affinity matrix, and the samples analyzed. Here, we attempt such a comparative study exploring a very large synthetic library containing thousands of serine, threonine, and tyrosine phosphorylated peptides, being present in roughly equal abundance, along with their nonphosphorylated counterparts, and use an optimized protocol for enrichment by TiO2 and Ti4+-irrunobilized metal affinity chromatography (IMAC) by a single operator. Surprisingly, our data reveal that there are minimal differences between enrichment of phosphopeptides by TiO2 and Ti4+-IIvLAC when considering biochemical and biophysical parameters such as peptide length, sequence surrounding the site, hydrophobicity, and nature of the amino acid phosphorylated. Similar results were obtained when evaluating a tryptic digest of a cellular lysate, representing a more natural source of phosphopeptides. All the data presented are available via ProteomeXchange with the identifier PXD000759.