Rapid Enrichment of Phosphopeptides and Phosphoproteins from Complex Samples Using Magnetic Particles Coated with Alumina as the Concentrating Probes for MALDI MS Analysis

In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to
selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution.
Tryptic digest products of phosphoproteins including α- and β-caseins, human protein phosphatase
inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrate the
feasibility of this approach. In only 30 and 90 s, phosphopeptides and phosphoproteins sufficient for
characterization by MALDI-MS were enriched by the particles, respectively. Proteins trapped on the
particles could be directly digested on the particles. The same particles in the digest solution were
employed for enrichment of phosphopeptides. We estimated the required time for performing the
enrichment of phosphopeptides from complex samples and characterization by MALDI MS was within
5 min. A small volume (50 μL) and a low concentration (5 × 10-10 M) of tryptic digest product of a
phosphoprotein sample could be dramatically enriched and characterized using this approach.
Keywords: alumina • magnetic particles • phosphopeptides • phosphoproteins • MALDI MS