Analysis of Protein Post-Translational Modifications Using Dige-Based Proteomics
DeKroon et al. DIGE-based PTMs
Analysis of protein post-translational modifications using DIGE-based proteomics
Robert M. DeKroon, Jennifer B. Robinette, Cristina Osorio, Sun Yong Jeong, Eric Hamlett, Mihaela Mocanu and Oscar Alzate
Summary
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 influenced by myriad post-translational modifications (PTMs), including palmitoylation, ubiquitination, oxidation, and phosphorylation. In this chapter, we describe the use of DIGE to determine specific PTMs by introducing specific labels, or changes in pI and/or molecular weight.
Key words: Phosphorylation, oxidation, ubiquitination, palmitoylation, post-translational modification, DIGE, neuroproteomics.
Corresponding Author: Oscar Alzate, Ph.D. 438A Taylor Hall, CB #7090, 104 Mason Farm Road. Chapel Hill, NC. 27599. Telephone: +1 (919) 962-3698. Email: alzate@email.unc.edu
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DeKroon et al. DIGE-based PTMs
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 post-translational modifications (PTMs). Therefore, it is important to determine changes in PTM levels in conjunction with protein expression. 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
Analysis of protein post-translational modifications using DIGE-based proteomics
Robert M. DeKroon, Jennifer B. Robinette, Cristina Osorio, Sun Yong Jeong, Eric Hamlett, Mihaela Mocanu and Oscar Alzate
Summary
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 influenced by myriad post-translational modifications (PTMs), including palmitoylation, ubiquitination, oxidation, and phosphorylation. In this chapter, we describe the use of DIGE to determine specific PTMs by introducing specific labels, or changes in pI and/or molecular weight.
Key words: Phosphorylation, oxidation, ubiquitination, palmitoylation, post-translational modification, DIGE, neuroproteomics.
Corresponding Author: Oscar Alzate, Ph.D. 438A Taylor Hall, CB #7090, 104 Mason Farm Road. Chapel Hill, NC. 27599. Telephone: +1 (919) 962-3698. Email: alzate@email.unc.edu
1
DeKroon et al. DIGE-based PTMs
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 post-translational modifications (PTMs). Therefore, it is important to determine changes in PTM levels in conjunction with protein expression. 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
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