Pilot investigation of magnetic nanoparticle-based immobilized metal affinity chromatography for efficient enrichment of phosphoproteoforms for mass spectrometry-based top-down proteomics

Abstract

Protein phosphorylation is a vital and common post-translational modification (PTM) in cells, modulating various biological processes and diseases. Comprehensive top-down proteomics of phosphorylated proteoforms (phosphoproteoforms) in cells and tissues is essential for a better understanding of the roles of protein phosphorylation in fundamental biological processes and diseases. Mass spectrometry (MS)-based top-down proteomics of phosphoproteoforms remains challenging due to their relatively low abundance. Herein, we investigated magnetic nanoparticle-based immobilized metal affinity chromatography (IMAC, Ti⁴⁺, and Fe³⁺) for selective enrichment of phosphoproteoforms for MS-based top-down proteomics. The IMAC method achieved reproducible and highly efficient enrichment of phosphoproteoforms from simple and complex protein mixtures. It outperformed one commercial phosphoprotein enrichment kit regarding the capture efficiency and recovery of phosphoproteins. Reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) analyses of yeast cell lysates after IMAC (Ti⁴⁺ or Fe³⁺) enrichment produced roughly 100% more phosphoproteoform identifications compared to without IMAC enrichment. Importantly, the phosphoproteoforms identified after Ti⁴⁺-IMAC or Fe³⁺-IMAC enrichment correspond to proteins with much lower overall abundance compared to that identified without the IMAC treatment. We also revealed that Ti⁴⁺-IMAC and Fe³⁺-IMAC could enrich different pools of phosphoproteoforms from complex proteomes and the combination of those two methods will be useful for further improving the phosphoproteoform coverage from complex samples. The results clearly demonstrate the value of our magnetic nanoparticle-based Ti⁴⁺-IMAC and Fe³⁺-IMAC for advancing top-down MS characterization of phosphoproteoforms in complex biological systems.

Keywords: Immobilized metal affinity chromatography; Magnetic nanoparticle; Phosphoproteoform; RPLC-MS/MS; Top-down proteomics.