Specificity of Immobilized Metal Affinity-Based IMAC/C18 Tip Enrichment of Phosphopeptides for Protein Phosphorylation Analysis

We have developed a simple, highly specific enrichment
procedure for phosphopeptides, by increasing the specificity of an immobilized metal affinity column (IMAC)
without using any chemical reaction. The method employs
a biphasic IMAC-C18 tip, in which IMAC beads are
packed on an Empore C18 disk in a 200-μL pipet tip.
Phosphopeptides are separated from non-phosphopeptides on the IMAC in an optimized solvent without any
chemical reaction, then desorbed from the IMAC using a
phosphate buffer, reconcentrated, and desalted on the
C18 disk. The increase in selectivity was achieved by (a)
using a strong acid to discriminate phosphates from
carboxyl groups of peptides and (b) using a high concentration of acetonitrile to remove hydrophobic non-phosphopeptides. The entire procedure was optimized by using
known phosphoproteins such as Akt1 kinase and protein
kinase A. Although it was difficult to detect phosphopeptides in MALDI-MS spectra of tryptic peptide mixtures
before enrichment, after the IMAC procedure, we could
successfully detect phosphopeptides with almost no non-phosphopeptides. Next, we constructed an array of IMAC−IMAC/C18 tips, such that number of arrayed tips on a
96-well plate could easily be changed depending on the
loading amount of sample. Applying this approach to
mouse forebrain resulted in the identification of 162
phosphopeptides (166 phosphorylation sites) from 135
proteins using nano-LC/MS.