Development of an off-line capillary column IMAC phosphopeptide enrichment method for label-free phosphorylation relative quantification.

Immobilized metal affinity chromatography (IMAC) and metal oxide type affinity chromatography (MOAC) techniques have been widely used for mass spectrometry-based phosphorylation analysis. Unlike MOAC techniques, IMAC requires rather complete removals of buffering reagents, salts and high concentrations of denaturant prior to sample loading in order for the successful enrichment of phosphopeptides. In this study, a simple off-line capillary column-based IMAC phosphopeptide enrichment method can shorten sample preparation time by eliminating the speed-vac step from the desalting process. Tryptic digest peptide samples containing 2M urea can be directly processed and the entire IMAC procedure can be completed within 6 h. When tryptic digest peptide samples prepared from mouse whole brain tissues were analyzed using our method, an average of 249 phosphoproteins and 463 unique phosphopeptides were identified from single 2-h RPLC-MS/MS analysis (~88% specificity). An additional advantage of this method is the significantly improved reproducibility of the phosphopeptide enrichment results. When four independent phosphopeptide enrichment experiments were carried out, the peak areas of phosphopeptides identified among four enrichment experiments were relatively similar (less than 16.2% relative standard dev.). Because of this increased reproducibility, relative phosphorylation quantification analysis of major phosphoproteins appears to be feasible without the need for stable isotope labeling techniques.