Refinement of an Optimized Batchwise IMAC Protocol
Konopka, David A.
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Protein phosphorylation is involved, either directly or indirectly, in every important cellular event. • Due to their minute concentrations, phosphopeptide samples must be enriched before they can be analyzed by mass spectrometry. • Immobilized metal affinity chromatography (IMAC) uses an immobilized metal cation (often Fe3+) to preferentially bind the phosphate group of a phosphopeptide (Figure 1). • Unfortunately, the current IMAC protocols, which call for samples to be run through consecutive IMAC and C18 columns, suffer from several drawbacks, most notably their lack of a high throughput capacity. The development of a batchwise IMAC protocol would solve these problems. • In a batchwise IMAC protocol, enrichment takes place in a microcentrifuge tube instead of a column, and C18 beads are not used. Since a batchwise protocol is designed for high throughput, a slightly higher sample loss is acceptable. • Lee et al. (2007) have developed a batchwise IMAC protocol, but it requires further refinement. • We theorized that their optimized wash buffer, which contains acetic acid (HOAc), would cause premature elution of bound phosphopeptides through interaction with the imido-diacetate (IDA) linkages immobilizing the Fe3+ on the beads. We also theorized that their method of elution, using a very acidic solution, would mobilize the Fe3+, allowing it to flow out with the eluate and impair later analysis of the phosphopeptides.