Optimized IMAC−IMAC Protocol for Phosphopeptide Recovery from Complex Biological Samples

Abstract

Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)-nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile-water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)-NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 microg of mouse sample or 50 microg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC-IMAC and LC-MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC-IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects.

Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

“…S1B), demonstrating that the use of an Fe-IMAC column solved the issue of capacity without compromising selectivity. As a result, we did not observe any overrepresentation of multiply phosphorylated peptides relative to other methods (8% in this cell line; Table I), which would be expected to occur if the capacity of the enrichment material were exceeded (15,46). The reproducibility of the Fe-IMAC column format was evaluated through analysis and quantification of phosphopeptides from three independent Fe-IMAC enrichments from the same A431 cell lysate digest.…”

Phosphosite Mapping of P-type Plasma Membrane H+-ATPase in Homologous and Heterologous Environments

“…Phosphopeptide Enrichment by IMAC-Peptide mixtures from 100 g of plant plasma membrane proteins were desalted with a Poros R3 column, and phosphopeptide enrichment was performed using IMAC as described (54).…”

Investigation of Receptor interacting protein (RIP3)-dependent Protein Phosphorylation by Quantitative Phosphoproteomics

“…Finally, the resins were resuspended in 0.1% acetic acid as a 50% (v/v) slurry, stored at 4°C and ready for IMAC enrichment. The IMAC procedure was performed according to optimized IMAC-IMAC protocol with minor modifications (30). The tryptic peptide samples corresponding to the 5 mg protein samples were re-dissolved in 1% acetic acid/60% acetonitrile, and incubated with a 40 l bed volume of IMAC beads.…”