Multiplex proteomic analysis by two‐dimensional differential in‐gel electrophoresis

Abstract

This paper describes the use of fluorescence two-dimensional differential in-gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin1 receptor knockout (NK 1 R2/2) and wild type (NK 1 R1/1) mice in an attempt to identify molecular pathways involved in the function of this protein. Paired NK 1 R2/2 and NK 1 R1/1 samples were labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed on the same two-dimensional gels. Scanning the gels at wavelengths specific for each dye revealed the two different proteomes which were overlaid and the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In-gel Analysis software. A Cy2-labeled sample pool was co-electrophoresed with all Cy3-and Cy5-labeled sample pairs as an internal standard providing a link for inter-gel comparisons and for more robust statistical analysis of the data. Eight spots were found to be upregulated and two downregulated in the NK 1 R2/2 mice compared to NK 1 R1/1 controls. Matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass fingerprinting was used to identify the proteins. The results illustrate the power of this multiplex proteomics technology and illustrate how proteomics can be used to understand gene function.

Cytosolic proteomic alterations in the nucleus accumbens of cocaine overdose victims

“…Figure 5a is representative of protein identification in this study. The GFAP identification was validated by sequencing two peptides, ALAAELNQLR [94][95][96][97][98][99][100][101][102][103] and LADVYQAELR, [110][111][112][113][114][115][116][117][118][119] having precursor ion m/z of 1098.6 and 1177.6, respectively (Figure 5b and c). Some of the protein spots that yielded significant DeCyder ratios were not identified with MALDI-ToF-MS, which can be attributed to insufficient amounts of protein.…”

“…The accuracy of quantitation as well as the statistical confidence obtained for the differentially regulated proteins is significantly higher using the experimental design of 2D-DIGE. 47,63,117,118 Moreover, 2D-DIGE offers the most reliable quantitation of any twodimensional gel electrophoresis (2DGE) method, is comparable in sensitivity to the silver staining method, yet is compatible with the downstream mass spectrometry protein characterization (as majority of the lysine residues remain untagged and accessible for tryptic digestion). An important caveat of the labeling method is that proteins with a high percentage of lysine residues are likely labeled more efficiently compared with the proteins with few/no lysine residues.…”

Protein expression profiling identifies maspin and stathmin as potential biomarkers of adenoid cystic carcinoma of the salivary glands

“…The technique provides reliable identification of proteomic differences between the samples, because multiple samples can be analyzed on a single gel and an internal standard can be included for accurate matching across gels. 22 The use of an internal standard helps to minimize false positives and negatives, because it provides separate control for each individual protein spot on all gels in the analysis. 22 It is possible that the CyDyes may show some artifactual preferential labeling of some proteins, although we did not detect any.…”

“…22 The use of an internal standard helps to minimize false positives and negatives, because it provides separate control for each individual protein spot on all gels in the analysis. 22 It is possible that the CyDyes may show some artifactual preferential labeling of some proteins, although we did not detect any.…”

Towards the proteome of the marine bacteriumRhodopirellula baltica: Mapping the soluble proteins

“…Quantitative resolution of changes in protein patterns by 2-DE requires reduction of gel-to-gel variations and sensitive protein dyes with a broad dynamic range. The 2-D DIGE technology, which is based on covalent labeling of proteins with fluorescent dyes prior to electrophoresis and application of an internal standard, allows determination of differences in protein abundance with statistical confidence [8][9][10][11].…”