2D Gel Proteomics

Abstract

This chapter describes protocols for two-dimensional (2D) gel electrophoresis (isoelectric focusing [IEF] followed by sodium-dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis [PAGE]), staining of gels with the fluorescent dye Sypro Ruby, 2D gel image analysis, peptide mass fingerprint (PMF) analysis using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS), liquid chromatography (LC)-tandem mass spectrometry (MS/MS), Western blot analysis of protein oxidations, and mass spectrometric mapping of sites of protein oxidations. Many of these methods were used to identify proteins affected in rat brain following ingestion of grape seed extract (GSE), a dietary supplement touted for anti-oxidant activity. Although beneficial actions in cell and animal models of chronic disease have been described for GSE, it has not been shown whether specific proteins were affected, or the nature of the effects. Applying 2D gel proteomics technology allowed discovery of proteins targeted by GSE without a priori knowledge of which one(s) might be affected. The newer 2D blue native (BN) electrophoresis methodology, which resolves protein complexes in a nondenaturing first dimension and then the components of these complexes in a denaturing second dimension, is discussed as a complementary approach. Analysis of protein oxidations and protein-protein interactions have special relevance to aging-related research, since oxidative stress and altered protein interactions may be at the heart of aging-related diseases. Finally, quality control issues related to implementation of high throughput technologies are addressed, to underscore the importance of minimizing bias and randomizing human and technical error in generating large datasets that are expensive and time-consuming to repeat.

FBXW7 influences murine intestinal homeostasis and cancer, targeting Notch, Jun, and DEK for degradation

“…4, A vs. C) and nuclear (Fig. 4, E vs. G) protein extracts using two-dimensional matrix-assisted laser desorption/ionization (MALDI)–mass spectrometry (MS) assays (Kim et al, 2007). Subsequent amino acid sequencing and bioinformatic analyses identified several proteins known to be involved in carcinogenesis.…”

“…Intestinal samples from young (≤5 wk) mutant Fbxw7 ΔG and control Fbxw7 fl/fl mice were initially homogenized with ice-cold PBS, and protein extracts were derived following fractionation cytosolic and nuclear extracts according to the manufacturer’s instructions (BioVision), resuspended in two-dimensional lysis buffer (Kim et al, 2007), and loaded separately onto Immobiline DryStrip gels containing a preformed pH gradient (pH 3–10), and proteins were run on the Protean IEF Cell (Bio-Rad Laboratories) according to the manufacturer’s instructions, separated on a 12% polyacrylamide gel, and then stained with Coomassie blue. Gels were scanned on a calibrated imaging densitometer (GS-800; Bio-Rad Laboratories), and images were analyzed using PDQuest (Bio-Rad Laboratories).…”

Delineation of Concentration Ranges and Longitudinal Changes of Human Plasma Protein Variants

“…One-dimensional gel isoelectric focusing was the method of choice, contributing to the discovery of variants for proteins such as hemoglobin [5], alpha-antitrypsin [6], amylase [7], and prealbumin (transthyretin) [8]. Subsequently, two-dimensional gel electrophoresis provided increased resolving power for simultaneous analysis of hundreds of proteins and their variants, especially when coupled to mass spectrometric (MS) identification of the protein spots from the gel [9]. Two-dimensional difference gel electrophoresis (DIGE) has been used in delineating protein variations across populations, as shown in the study of longitudinal and individual variation of 78 proteins from eleven healthy subjects [10], the normal variability of the human plasma proteome from 60 healthy subjects [11], and age-related differences in 100 plasma proteins from 42 individuals [12].…”

Proteomics and Cardiovascular Disease: An Update

“…teomics [20,[35][36][37] as it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or posttranslational modifications [38,39].…”