Pyridoxal 5'-phosphate mediated preparation of immobilized metal affinity material for highly selective and sensitive enrichment of phosphopeptides

Abstract

Phosphorylation is a crucial post-translational modification, which plays pivotal roles in various biological processes. Analysis of phosphopeptides by mass spectrometry (MS) is intractable on account of their low stoichiometry and the ion suppression from non-phosphopeptides. Thus, enrichment of phosphopeptides before MS analysis is indispensable. In this work, we employed pyridoxal 5'-phosphate (PLP), as an immobilized metal affinity chromatography (IMAC) ligand for the enrichment of phosphopeptides. PLP was grafted onto several substrates such as silica (SiO₂), oxidized carbon nanotube (OCNT) and silica coated magnetic nanoparticles (Fe₃O₄@SiO₂). Then the metal ions Fe³⁺, Ga³⁺ and Ti⁴⁺ were incorporated for the selective enrichment of phosphopeptides. It is indicated that Fe₃O₄@SiO₂-PLP-Ti⁴⁺ has a superior selectivity towards phosphopeptides under as much as 1000-fold interferences of non-phosphopeptides. Further, Fe₃O₄@SiO₂-PLP-Ti⁴⁺ exhibited high efficiency in selective enrichments of phosphopeptides from complex biological samples, including human serum and tryptic digested non-fat milk. Finally, Fe₃O₄@SiO₂-PLP-Ti⁴⁺ was successfully employed in the sample pretreatment for profiling phosphopeptides in a tryptic digest of rat brain proteins. Our experimental results evidenced a great potential of this new chelator-based material in phosphoproteomics study.

Keywords: Immobilized metal affinity chromatography; Phosphopeptide enrichment; Pyridoxal 5′-phosphate; Rat brain.