Refinement of an Optimized Batchwise IMAC Protocol

dc.contributor.authorKonopka, David A.
dc.description.abstractProtein 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.en
dc.description.sponsorshipKalamazoo College. Department of Biology. Diebold Symposium, 2008.
dc.publisherKalamazoo, Mich. : Kalamazoo College.
dc.relation.ispartofKalamazoo College Diebold Symposium Presentations Collection
dc.rightsU.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.
dc.titleRefinement of an Optimized Batchwise IMAC Protocolen