Abstract
Recent developments in first dimension High-Performance Liquid Chromatography (HPLC) separation of complex peptide mixtures, followed by a subsequent immobilized metal ion affinity chromatography (IMAC) for phosphopeptide enrichment have shown great promise in both selectivity and quantification of phosphopeptides via LC-MS/MS analysis. The first dimension HPLC, such as hydrophilic interaction chromatography (HILIC) or high pH Reverse Phase chromatography, was employed for its being orthogonal to the second dimension chromatography gradient and/or its even distribution of phosphopeptides among fractions derived from separation. Subsequent IMAC enrichment can then achieve high specificity and superb quantification for SILAC LC-MS/MS approaches. However, first dimension HPLC separation can generate a large number of fractions, each of which is handled in a microtube, such as a Nylon filter tube or a stage tip device, for the following IMAC procedure. Handling so many tubes will make the IMAC procedure not only more tedious but also reduce reproducibility when biological replicates are required. In our current improved IMAC protocol, we employed a 96 well glass fiber plate which replaces both the individual nylon filter tubes from the HILIC-IMAC workflow and cumbersome stage tips in the RP-IMAC workflow. Our results indicate that we can identify ∼20,000 unique phosphopeptides with 3mg of mouse brain digest input with specificity above 90%. In addition, we are able to overcome contaminating nylon filter peaks in the HILIC-IMAC workflow. Our IMAC protocol significantly shortens the IMAC method to ∼1hr instead of several hours with minimum handling of individual microtubes and/or stage tips. In conclusion, our newly improved IMAC phosphopeptide enrichment method achieves a fast and effective phosphopeptide enrichment workflow that retains high specificity and great quantification, therefore providing a very useful tool for global phosphoproteomics studies.