Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE)

Abstract

Two-dimensional gel electrophoresis (2-D GE) is a key tool for comparative proteomics research. With its ability to separate complex protein mixtures with high resolution, 2-D GE is a technique commonly employed for protein profiling studies. Significant improvements have been made in 2-D GE technology with the development of two-dimensional fluorescence difference gel electrophoresis (2-D DIGE), where proteins are first labelled with one of three spectrally resolvable fluorescent cyanine dyes before being separated over first and second dimensions according to their charge and size, respectively. When used in conjunction with automated analysis packages, this multiplexing approach can accurately and reproducibly quantify protein expression for control and experimental groups. Differentially expressed proteins can be subsequently identified by mass spectrometric methods. Here, we describe the successful application and optimisation of 2-D DIGE technology for human postmortem brain studies. This technology, especially when coupled with other functional genomics approaches, such as transcriptomics and metabolomics studies, will enhance our current understanding of human disease and lead to new therapeutic and diagnostic possibilities.

Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims

“…46,[48][49][50][51] Such studies have confirmed the differential abundance of certain proteins and provided refined hypotheses regarding the molecular mechanisms of these diseases. Two-dimensional electrophoresis (2DE), which separates protein species by both isoelectric point and molecular weight, 52 has been widely used and recently been improved through the addition of direct labeling of proteins with cyanine (Cy) dyes to facilitate quantitation (two-dimensional difference gel electrophoresis (2D-DIGE)).…”

“…The average ratio (COD:control (n = 10)) as well as the corresponding Student's t-test value for each protein spot was calculated based on all gel images in the DeCyder BVA mode. 50 The protein of interest based on statistically significant t-test was further assessed by the three-dimensional spot profile to validate the spot on most of the gel images.…”

“…71 Gel plugs of 2 mm size were cut robotically using the Ettan Spot Handling Workstation (GE Healthcare). 50 Picked proteins were prepared for mass spectrometry analysis using a standard in-gel trypsin digestion protocol. Briefly, the gel plugs were washed for 20 min, twice in 50 mM ammonium bicarbonate/50% (v/v) methanol in water and once with 75% (v/v) acetonitrile (ACN) in DW for 30 min or until the gel plugs turn opaque.…”

“…The selection of specific pH range used in the present study for separation of proteins in the first dimension was determined empirically and was based on the pH range that provided optimal resolution of proteins present in the complex mixture in question. Previous studies have utilized broad-range pH strips 46,49,50 as well as more narrow-range pH strips 51,55,56,72 for a range of complex protein samples. For complex protein mixtures, it has been noted that the majority of the protein spots resolved using 3-10 pH strip usually fall between 4 and 7 pH.…”

Is protein overlap in two‐dimensional gels a serious practical problem?

“…To suggest that this is cause for "serious concern" is an overreaction. There are numerous published reports of the utility of 2-D gels for relative quantification of proteins, particularly when DIGE is employed [9][10][11][12][13] and these stand as testimony to the power of the 2-D gel strategy. Improvements in detection methods will continue for the foreseeable future and will reveal that our samples are more complex than prior work indicated.…”