Development of amphiphile 4-armed PEO-based Ti4+ complex for highly selective enrichment of phosphopeptides.

Protein phosphorylation is a reversible and important post-translational modification. Identification of phosphopeptides without enrichment is difficult for the low-abundance of phosphopeptides in real complex biological samples. Therefore, the effective and selective concentration of phosphopeptides prior to proteomic identification by mass spectrometer is necessary. In this study, we synthesized a novel titanium-based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. To improve material hydrophilia to the maximum extent, titanium ions were immobilized on the 4-armed Poly(ethylene oxide)(4µ-PEO-Ti4+), a totally soluble polymer with large molecular weight (20000 g/mol). The 4µ-PEO-Ti4+ was used to enrich phosphopeptides from tryptic digests of standard proteins and real complex biological samples, followed by MALDI-TOF MS analysis. In enrichment of phosphopeptides from 4 pmol ß-casein, the 4µ-PEO-Ti4+ performed the best property with starting material of 99-132 µg, loading buffer of 50% ACN/5% TFA (v/v), elution buffer of 10% NH3·H2O (v/v) and elution time of 30 min. The 4µ-PEO-Ti4+ has a superior detection sensitivity as low as 2 fmol for phosphopeptides. The high selectivity of 4µ-PEO-Ti4+ allows a deep enrichment of phosphopeptides of ß-casein from a mixture with BSA of 1000-fold abundant. The 4µ-PEO-Ti4+ shows great stability and endurability and can be recycled up to at least 5 times. In addition, 4µ-PEO-Ti4+ could detect 10 and 15 phosphopeptides from non-fat milk and nonenzymatic human saliva, respectively. In total, 4µ-PEO-Ti4+ is a novel excellent material which shows great sensitive and selective enrichment of low-abundance phosphopeptides in real complex biological samples.