De novo protein sequence analysis of Macaca mulatta.

Background: Macaca mulatta is one of the most utilized non-human primate species in biomedical research offering unique behavioral, neuroanatomical, and neurobiochemcial similarities to humans. This makes it a unique organism to model various diseases such as psychiatric and neurodegenerative illnesses while also providing insight into the complexities of the primate brain. A major obstacle in utilizing rhesus monkey models for human disease is the paucity of protein annotations for this species (~42,000 protein annotations) compared to 330,210 protein annotations for humans.

Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling.

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).

Methods for proteomics in neuroscience.

Proteomics reveals complex protein expression, function, interactions and localization in different phenotypes of neuron. As proteomics, regarded as a highly complex screening technology, moves from a theoretical approach to practical reality, neuroscientists have to determine the most-appropriate applications for this technology. Even though proteomics compliments genomics, it is in sheer contrast to the basically constant genome due to its dynamic nature.

Effect of staining reagent on peptide mass fingerprinting from in-gel trypsin digestions: a comparison of SyproRuby and DeepPurple.

As the new fluorescent stains such as SyproRuby and DeepPurple are getting widespread recognition for proteome analyses by the traditional 2-D gel method, it becomes important to test the feasibility of these stains with respect to staining reproducibility, protein quantitation, and compatibility of the stain with downstream MS. The binding of epicocconone, active ingredient of DeepPurple, to one of the primary cleavage sites of trypsin (lysine residue) raises the possibility of incomplete cleavage and interference with PMF. However, the current study tests and concludes that the DeepPurple stain can result in increased peptide recovery compared to SyproRuby stain and can improve MS-based identification of lower intensity proteins spots.

Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program.

When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined 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.

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting.

We compared trysin-digested protein samples desalted by ZipTip(C18) reverse-phase microcolumns with on-plate washing of peptides deposited either on paraffin-coated plates (PCP), Teflon-based AnchorChip plates, or stainless steel plates, before analysis by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Trypsinized bovine serum albumin and ovalbumin and 16 protein spots extracted from silver-stained two-dimensional gels of murine C(2)C(12) myoblasts or human leukocytes, prepared by the above two methods, were subjected to MALDI on PCP, AnchorChip plates, or uncoated stainless steel plates. Although most peptide mass peaks were identical regardless of the method of desalting and concentrating of protein samples, samples washed and concentrated by the PCP-based method had peptide peaks that were not seen in the samples prepared using the ZipTip(C18) columns.