Quantitative proteomics is the workhorse of the modern proteomics initiative. The gel-based and MuDPIT approaches have facilitated vital advances in the measurement of protein expression alterations in normal and disease phenotypic states. The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). 2D-DIGE is based on direct labeling of lysine groups on proteins with cyanine CyDye DIGE Fluor minimal dyes before isoelectric focusing, enabling the labeling of 2-3 samples with different dyes and electrophoresis of all the samples on the same 2D gel. This capability minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching. This protocol can be completed in 3-5 weeks depending on the sample size of the experiment and the level of expertise of the investigator.

When cultured in low serum-containing growth medium, the mouse C 2 C 12 cells exit cell cycle and undergo a welldefined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane-and nuclear-enriched proteins, and phospho-proteins from the proliferating C 2 C 12 cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C 2 C 12 myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C 2 C 12 myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phosphoproteins also occurred during conversion of C 2 C 12 myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESIquadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate interand intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation. Molecular & Cellular

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Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in neuronal function in the nucleus accumbens (NAc), a brain region associated with drug reinforcement. Two-dimensional gel electrophoresis was used to compare protein alterations in the NAc between cocaine overdose (COD) victims (n = 10) and controls (n = 10). Following image normalization, spots with significantly differential image intensities (P < 0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser desorption ionization-time of flight-time of flight. A total of 1407 spots were found to be present in a minimum of five subjects per group and the intensity of 18 spots was found to be differentially abundant between the groups, leading to positive identification of 15 proteins by peptide mass fingerprinting (PMF). Of an additional 37 protein spots that were constitutively expressed, 32 proteins were positively identified by PMF. Increased proteins in COD included b-tubulin, liprin-a3 and neuronal enolase, whereas decreased proteins included parvalbumin, ATP synthase b-chain and peroxiredoxin 2. The present data provide a preliminary protein profile of COD, suggesting the involvement of novel proteins and pathways in the expression of this complex disease. Additional studies are warranted to further characterize alterations in the differentially regulated proteins. Understanding the coordinated involvement of multiple proteins in cocaine abuse provides insight into the molecular basis of the disease and offers new targets for pharmacotherapeutic intervention for drug abuse-related disorders.

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