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

Abstract

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-microL 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.

Hydrophilic Interaction Chromatography Reduces the Complexity of the Phosphoproteome and Improves Global Phosphopeptide Isolation and Detection

“…Second, the acetonitrile/water solvent system for the TSKgel Amide-80 column contains 0.1% TFA, and the majority of phosphopeptides elute between 70 and 50% acetonitrile. Oda and co-workers (22) have shown that the best recovery and greatest selectivity for IMAC occurs when the loading buffer contains between 0.1 and 1.0% TFA and between 45 and 65% acetonitrile. Thus FIG.…”

G Protein–Coupled Receptor Endocytosis Confers Uniformity in Responses to Chemically Distinct Ligands

“…Phosphopeptides were purified from approximately 1 mg each sample. The use of IMAC/C18 columns is described in the literature (Kokubu et al, 2005;Ficarro et al, 2009;Mertins et al, 2013). Fe 31 -IMAC resin was created by taking Ni-NTA Superflow agarose resin (Qiagen, Germantown, MD) and stripping out Ni by incubating with 500 mM EDTA, pH 8 (1:1 v/v), for 5 minutes, and repeating.…”

Analytical strategies for phosphoproteomics

“…By decrease in the pH of the IMAC loading conditions further (below pH 1.9), more acidic peptides will become neutralized while phosphopeptides will retain their negative charge and their binding affinity toward the IMAC resin. Kokubu and co-workers showed that 0.1% TFA, 50% ACN as IMAC loading buffer reduced the level of nonspecific binding and improved the specificity of the method toward phosphopeptides [56]. Yet another approach to improve the performance of IMAC is to reduce the complexity of the peptide samples by prefractionation using methods such as IEF [57][58][59][60], Ion Exchange Chromatography [41,43,46,61] or hydrophilic interaction chromatography (HILIC) [62] prior to IMAC purification.…”