Refinement of an Optimized Batchwise IMAC Protocol

Abstract

Phosphorylation plays a key role in regulating metabolic activities in cells. The study of phosphorylated proteins is currently centered around their digestion into phosphopeptides and subsequent analysis. However, due to the very low concentration of proteins that are phosphorylated at any given time, samples must be enriched prior to analysis by mass spectrometry. Immobilized metal (Fe3+) affinity chromatography (IMAC) is, at present, the most promising enrichment technique available. Unfortunately, the current column-based technique is relatively slow, is incapable of using organic buffers, and does not have a high throughput capacity. Developing an optimized batchwise IMAC protocol, like that of Lee et al. (2007), would resolve these issues, but refinements of the wash buffer and elution method are still needed. We used a mixture of tryptically digested b-casein, a common phosphoprotein, and a phosphopeptide standard to test the batchwise IMAC protocol with different wash and elution buffers. We were able to analyze the results quantitatively by comparing the total ion content of the samples, obtained by tandem mass spectrometry. The data suggest that there is a slight improvement in signal strength when decreasing the HOAc content of the wash buffer. Importantly, there also appears to be a large improvement when eluting the phosphopeptides by phosphate exchange instead of with a very acidic solution. These . data suggest that signal strength can be greatly enhanced through simple changes to the buffers used. Moreover, they demonstrate that is important to look not only at the elution of phosphopeptides off of the beads, but also at the elution of phosphopeptides off of the Fe 3 + .