Biology Reference
In-Depth Information
Chapter 9
Analysis of Protein Posttranslational Modifi cations
Using DIGE-Based Proteomics
Robert M. DeKroon , Jennifer B. Robinette , Cristina Osorio ,
Joseph S. Y. Jeong , Eric Hamlett , Mihaela Mocanu , and Oscar Alzate
Abstract
Difference gel electrophoresis (DIGE) is most often used to assess relative changes in the expression levels of
individual proteins in multiple complex samples, and this information is valuable in making inferences about
relative protein activity. However, a protein's activity is not solely dependent upon its expression level.
A change in activity may also be infl uenced by myriad posttranslational modifi cations (PTMs), including
palmitoylation, ubiquitination, oxidation, and phosphorylation. In this chapter, we describe the use of DIGE
to determine specifi c PTMs by introducing specifi c labels or changes in p
I
and/or molecular weight.
Key words:
Difference gel electrophoresis, Neuroproteomics, Oxidation, Palmitoylation, Phosphory-
lation, Posttranslational modifi cation, Ubiquitination
1. Introduction
In eukaryotic systems, a protein's expression level is a widely
accepted determination of its activity. However, protein activity is
further regulated by posttranslational modifi cations (PTMs).
Therefore, it is important to determine changes in PTM levels in
conjunction with protein expression. Difference gel electrophoresis
(DIGE) involves resolving proteins from multiple complex samples
within the same gel and is thus a valuable technique for assessing
relative protein expression. The high resolution of DIGE makes it
possible to distinguish PTMs if they introduce changes to pI and/
or relative mobility (Mr). Here, we present DIGE-based approaches
for identifying PTMs in multiple complex samples by exploiting
PTM-induced changes in pI and Mr. For example, by using phos-
phatases to remove the phosphate group from phosphorylated
tyrosine, serine, and threonine amino acid residues and comparing
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