By a News Reporter-Staff News Editor at Life Science Weekly -- Investigators discuss new findings in Peptides and Proteins. According to news reporting originating from Edmonton, Canada, by NewsRx correspondents, research stated, "We report an enabling method for mapping the protein sequence with high sequence coverage. This method combines the high separation power of gel electrophoresis for protein separation with the high sequence coverage capability of microwave-assisted acid hydrolysis (MAAH) mass spectrometry (MS)."
Our news editors obtained a quote from the research from the University of Alberta, "In-gel MAAH using 25% trifluoroacetic acid was developed and optimized for degrading the gel-separated protein into small peptides suitable for tandem MS sequencing. For bovine serum albumin (BSA) (similar to 67 kDa), with 4 mu g of protein loading onto a gel for separation, followed by excising the protein gel band for in-gel MAAH and then injecting similar to 2 mu g of the resultant peptides into a liquid chromatography quadrupole time-of-flight mass spectrometer for analysis, 689.+/- 54 (n = 3) unique peptides were identified with a protein sequence coverage of 99 +/- 1%. Both the number of peptides detected and sequence coverage decreased as the sample amount decreased, mainly due to background interference: 316 +/- 59 peptides and 94 + 3% coverage for 2 mu g loading, 136 +/- 19 and 76 +/- 5% for 1 mu g loading, and 30 +/- 2 and 32 +/- 2% for 0.5 mu g loading. To demonstrate the general applicability of the method, 10 gel bands from gel electrophoresis of an albumin-depleted human plasma sample were excised for in-gel MAAH LC-MS analysis. In total, 19 relatively high abundance proteins with molecular weights ranging from similar to 8 to similar to 160 kD could be mapped with coverage of 100% for six proteins (MW 8759 to 68 425 Da), 96-98% for five proteins (MW 11 458 to 36 431 Da), 92% for three proteins (MW 15971 to 36 431 Da), 80-87% for four proteins (MW 42 287 to 162 134 Da), and 56% for one protein (MW 51 358 Da). Finally, to demonstrate the applicability of the method for more detailed analysis of complex protein mixtures, two-dimensional (2D) gel electrophoresis was combined with in-gel MAAH, affinity purification, and LC-MS/MS to characterize six bovine alpha-S1-casein phosphoprotein isoforms."
According to the news editors, the research concluded: "Full sequence coverage was achieved for each protein, and six new modification sites were found."
For more information on this research see: In-Gel Microwave-Assisted Acid Hydrolysis of Proteins Combined with Liquid Chromatography Tandem Mass Spectrometry for Mapping Protein Sequences. Analytical Chemistry, 2014;86(1):600-607. Analytical Chemistry can be contacted at: Amer Chemical Soc, 1155 16TH St, NW, Washington, DC 20036, USA. (American Chemical Society - www.acs.org; Analytical Chemistry - www.pubs.acs.org/journal/ancham)
The news editors report that additional information may be obtained by contacting D.F. Sun, University of Alberta, Dept. of Chem, Edmonton, AB T6G 2G2, Canada. Additional authors for this research include N. Wang and L. Li (see also Peptides and Proteins).
Keywords for this news article include: Canada, Alberta, Edmonton, Proteomics, Amino Acids, Peptides and Proteins, North and Central America
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