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Genome Technology s Lab Notebook: Reader Tips and Experiences: Jan 13, 2005

Following are scientists' responses to the question, "How would you fractionate plasma or serum prior to mass spec analysis to increase the number of identified proteins?"

 

For a complete list of scientists' responses, read Genome Technology, a GenomeWeb News sister publication.


"The most successful approach in our hands has been one that combines complementary techniques both at the level of protein fractionation and peptide/protein identification by mass spectrometry. We commonly separate intact proteins by non-denaturing techniques followed by standard denaturing separation on one (1DE) or two (2DE) dimensional electrophoresis. 2DE is interfaced with MALDI-TOF peptide mass fingerprinting and LC-ESI/MS/MS, and 1D gels sequentially fractionated and analyzed by LC-ESI/MS/MS. Albumin and other abundant protein depletion by commercially available kits can be helpful to increase coverage of the proteome, and the most successful approaches will likely analyze the same sample both with and without depletion of abundant proteins."

 

Thomas M. Vondriska, PhD

Proteomics Laboratory

DavidGeffenSchoolof Medicine at UCLA


"Our approach is based on the use of specific chemical probes that can selectively tag and facilitate subsequent isolation of a target peptide of protein. Since most serum proteins are glycosylated, selective isolation of glycopeptides provides several advantages for serum protein analysis. First, the most abundant serum protein, albumin, does not contain N-linked glycosylation motifs and therefore is effectively transparent to the analysis. Second, many serum proteins are post-translationally altered by phosphorylation, glycosylation, acetylation, methionine oxidation, protease processing, and other mechanisms, resulting in multiple forms for each protein."

 

Hui Zhang, PhD

Institute for Systems Biology

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