title: Characterization of biases in phosphopeptide enrichment by Ti(4+)-immobilized metal affinity chromatography and TiO2 using a massive synthetic library and human cell digests.
abstract: 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 Ti(4+)-immobilized metal affinity chromatography (IMAC) by a single operator. Surprisingly, our data reveal that there are minimal differences between enrichment of phosphopeptides by TiO2 and Ti(4+)-IMAC 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.
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences and The Netherlands Proteomics Centre, Utrecht University , Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors: Lucrece
authors: L
authors: Matheron
authors: Henk
authors: H
authors: van den Toorn
authors: Albert J R
authors: AJ
authors: Heck
authors: Shabaz
authors: S
authors: Mohammed
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D010766:Phosphorylation; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry*
chemical_list: D019151:Peptide Library; D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium