Common Mass Spectrometry Based Proteomic Methodologies Used On Day Is The Bottom Up Protein Identification

824 Words Apr 8th, 2016 4 Pages
One of the most common mass spectrometry–based proteomic methodologies used to day is the bottom up protein identification. First, the protein has to be digested into peptides using a protease. Then, the peptide will be submitted to the URMC Proteomics Resource and Laboratory for analysis using LC-MS/MS tandem mass spectrometry. In this approach, the peptides will be separated and introduced into a mass spectrometer using reversed phase liquid chromatography (RC-LC). This spectrometer will measure the mass and charge of each peptide. Then, the peptide will be isolated and fragmented along the backbone to generate tandem of mass spectrum. The peptide MS/MS spectra will then be searched against a known protein database in order to identify the protein (1).
In order to analyze the protein using mass spectrometry, the protein has to be digested by a protease first. Protease plays in important role in processing the protein for proteomic analysis. Only a few proteases that are useful for proteomic analysis. For instance, trypsin and chymotrypsin are often used to cleave proteins at selective amino acid residues. Trypsin cleaves on the C-terminal side of the lysine and arginine amino acids, whereas chymotrypsin cleaves after the aromatic amino acids. Trypsin exhibits higher activity and narrower specificity compared to chymotrypsin. When the lysine reside is modified or flanked by proline, cleavage of trypsin is blocked (1). Since trypsin has higher activity, it…

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