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: [phone]. Email: [e-mail]

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

References: 1. Yamagata A, Kristensen DB, Takeda Y, Miyamoto Y, Okada K, Inamatsu M, Yoshizato K (2002) Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase. Proteomics 2: 1267-1276. 2. Sun J, Morgan M, Shen R-F, Steenbergen C and Murphy E (2007) Preconditioning Results in S-Nitrosylation of Proteins Involved in Regulation of Mitochondrial Energetics and Calcium Transport. Circ Res 101: 1155-1163. 4. Diez R, Herbstreith M, Osorio C, Alzate O (2009) 2-D Fluorescence Difference Gel Electrophoresis (DIGE) in neuroproteomics. In: Alzate O (ed) Neuroproteomics, CRC Press, Boca Raton 5. DeKroon RM, Osorio C, Robinette JB, Mocanu M, Winnik WM, Alzate O (2011). 6. Storey JD and Tibshirani R (2003) Statistical significance for genomewide studies. PNAS 100: 9440-9445. 7. Sanchez JC, Chiappe D, Converset V, Hoogland C, Binz PA, Paesano S, Appel RD, Wang S, Sennitt M, Nolan A, Cawthorne MA, Hochstrasser DF (2001) The mouse SWISS-2D PAGE database: a tool for proteomics study of diabetes and obesity. Proteomics 1: 136-163. 8. Zhoa YM, Basu U, Dodson MV, Basarab JA, Guan LL (2010) Proteome difference associated with fat accumulation in bovine subcutaneous adipose tissues. Proteome Sci 8: 1-14. 9. Babu GJ, Wheeler D, Alzate O, Periasamy M (2004) Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins. Anal Biochem 325: 121-125. 10. Blackstone CD, Moss SJ, Martin LJ, Levey AI, Price DL, Huganir RL (1992) Biochemical characterization and localization of a non-N-methyl-D-aspartate glutamate receptor in rat brain. J Neurochem 58: 1118-1126. 11. Lau LF, Mammen A, Ehlers MD, Kindler S, Chung WJ, Garner CC, Huganir RL (1996) Interaction of the N-methyl-D-aspartate receptor complex with a novel synapse-associated protein, SAP102. J Biol Chem 271: 21622-21628. 12. Wessel D and Flugge UI (1983) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138: 141-143.