Refinement of an Optimized Batchwise IMAC Protocol
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Authors
Konopka, David A.
Issue Date
2008
Type
Presentation
Language
en_US
Keywords
Alternative Title
Abstract
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.
Description
Citation
Publisher
Kalamazoo, Mich. : Kalamazoo College.
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.