Refinement of an Optimized Batchwise IMAC Protocol
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Authors
Konopka, David A.
Issue Date
2008
Type
Thesis
Language
en_US
Keywords
Alternative Title
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 + .
Description
vi, 18 p.
Citation
Publisher
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.