Enrichment of Phosphopeptides by Fe³⁺-Immobilized Magnetic Nanoparticles for Phosphoproteome Analysis of the Plasma Membrane of Mouse Liver

Immobilized metal ion affinity chromatography (IMAC) is a commonly used technique for phosphoprotein analysis due to its specific affinity for phosphopeptides. In this study, Fe³⁺-immobilized magnetic nanoparticles (Fe³⁺-IMAN) with an average diameter of 15 nm were synthesized and applied to enrich phosphopeptides. Compared with commercial microscale IMAC beads, Fe³⁺-IMAN has a larger surface area and better dispersibility in buffer solutions which improved the specific interaction with phosphopeptides. Using tryptic digests of the phosphoprotein α-casein as a model sample, the number and signal-to-noise ratios of the phosphopeptides identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following Fe³⁺-IMAN enrichment greatly increased relative to results obtained with direct MALDI-TOFMS analysis. The lowest detectable concentration is 5 × 10⁻¹¹ M for 100 µL of pure standard phosphopeptide (FLTEpYVATR) following Fe³⁺-IMAN enrichment. We presented a phosphopeptide enrichment scheme using simple Fe³⁺-IMAN and also a combined approach of strong cation exchange chromatography and Fe³⁺-IMAN for phosphoproteome analysis of the plasma membrane of mouse liver. In total, 217 unique phosphorylation sites corresponding to 158 phosphoproteins were identified by nano-LC-MS/MS. This efficient approach will be very useful in large-scale phosphoproteome analysis.