Highly selective enrichment of phosphopeptides by on-chip indium oxide functionalized magnetic nanoparticles coupled...

Selective and efficient preconcentration is indispensable for low concentration of phosphopeptides in phosphorylated protein-related samples prior to MS-based analysis. Herein, an on-chip system coupled magnetic SPE with MALDI-TOF MS was designed. A metal oxide affinity chromatography material, indium oxide, was coated on the surface of Fe₃ O₄ magnetic nanoparticles to prepare the adsorbent, spatially confined with an applied magnetic field. The adsorbent exhibited high selectivity for phosphopeptides in tryptic digests of the mixture of β-casein and BSA (1:1000) and the mixture of β-casein, BSA, and ovalbumin (1:100:100). Thanking to the enrichment ability and specificity for phosphopeptides with the adsorbent, the on-chip magnetic SPE-MALDI-TOF MS approach showed high sensitivity with a low detection limit of 4 fmol. In addition, the developed approach was used to analyze phosphopetides in non-fat milk digests and human serum successfully.