Facile preparation of monolithic immobilized metal affinity chromatography capillary columns for selective enrichment of phosphopeptides

Abstract

The selective enrichment of phosphopeptides with good reproducibility is vital for the in-depth study of the phosphoproteome. Herein, we presented a novel method to prepare monolithic Ti(4+) or Zr(4+) immobilized metal affinity chromatography (IMAC) columns. Since succinimide was of high reaction activity with nitrilotriacetic acid (NTA) than traditional epoxy group, through the reaction of succinimide group on the monolithic matrix with nitrilotriacetic acid, the preparation of monolithic IMAC columns became facile. By the developed new method, not only the time required for Ti(4+) or Zr(4+) immobilization could be shortened from 10 to 1 h, but also the RSD of obtained phosphopeptide peak area was below 13%, even with IMAC columns prepared in different batches (n=3). With such monolithic Ti(4+)- and Zr(4+)-IMAC columns, ten and seven phosphopeptides were effectively identified from the digests of a mixture of β-casein and BSA, even with the molar ratio as low as 1:100, respectively. The phosphopeptide recovery was over 73%, and the loading capacity was over 0.8 μmol/mL. Compared with commercial IMAC beads, the monolithic IMAC columns provide outstanding reproducibility, good selectivity, large loading capacity, and high recovery, which demonstrates that such monolithic IMAC columns might facilitate the in-depth analysis of phosphoproteomes.