Phosphopeptide enrichment with cross-linked Os(II)(dmebpy)₂ Cl-derivatized acrylamide and vinylimidazole copolymer

Reversible phosphorylation of proteins catalyzed by kinases and phosphatases plays a key regulatory role in intracellular biological processes. Protein phosphorylation profiling is still a challenge due to its low stoichiometry, diversity of phosphorylated protein forms, and dynamic nature of phosphorylation states. Mass spectrometry (MS) has been widely used for the characterization of protein phosphorylation, due to its high sensitivity and MS/MS sequencing capability. However, the low abundance and ionization efficiency of phosphorylated peptides and interference from their non-phosphorylated counterparts and other peptides in the enzymatic digests of proteins complicate the localization of phosphorylation sites in liquid chromatography (LC)/MS analysis. So the enrichment of phosphopeptides from the digests is often required before LC/MS. Immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) are the two most commonly used enrichment techniques for phosphopeptides prior to MS analysis.

Cross-linked Os(II)(4,4'-dimethyl-2,2'-bipyridine)₂ Cl-derivatized acrylamide and vinylimidazole copolymer were applied for the enrichment of phosphopeptides.

Under neutral loading buffer conditions phosphopeptides bind on the Os-polymer without nonspecific binding of acidic peptides. Differential binding of monophosphorylated and multiply phosphorylated peptides can be achieved under different concentrations of imidazole. Sequential elution of bound phosphopeptides can be obtained with elution buffers of different pH values below 3. The loading buffers with imidazole can be aqueous or 7/3 H₂ O/ACN. Once phosphopeptides bind onto the Os-polymer, washing with water, 0.1% acetic acid (pH ~ 3) or 1/1 H₂ O/ACN 0.05% acetic acid (pH ~3) does not elute phosphopeptides. The Os-polymer does not show bias of binding and elution toward phosphopeptide standards with singly, doubly and triply phosphorylated sites.

Cross-linked Os(II)(dimethylbipyridine)₂ Cl-derivatized poly(acrylamide)-poly(vinylimidazole) copolymer is proven to be a new efficient IMAC resin for phosphopeptide enrichment and shows some unique properties for differential binding and sequential elution of phosphopeptides. It could become a better alternative to traditional IMAC and TiO₂ for phosphopeptide enrichment.