ANYL 24 |
| Most proteomics methods take a discovery based approach by rapidly acquiring peptide fragmentation spectra using data-dependent µLC-MS/MS. These approaches now facilitate the identification of 1,000's of proteins in a single analysis in an unbiased fashion. Combined with biochemical and subcellular fractionation even low abundance proteins can be identified and quantified. However, these discovery based proteomics approaches are slow – often requiring a full day to weeks of instrument time for a single experiment to identify low abundance proteins. Additionally, the quantitative linear range in often limited to 10^1 - 10^2. An alternative approach is hypothesis driven and is the mass spectrometry equivalent of a Western blot. In this approach, a unique tryptic peptide(s) for a protein of interest is monitored by selected reaction monitoring (SRM) mass spectrometry, and the peptide precursor ion, fragment ion, and retention time facilitate the detection and quantitation of the protein in a mixture. Detection limits using SRM on a triple quadrupole mass spectrometer are presently at <10 attamole of peptide injected on column in the context of a complex mixture, and the quantitative linear dynamic range is an amazing 10^5. Analyses can be completed in 15-30 minutes with a fraction of the sample normally required for undirected experiments making multiple analyses possible in a short period of time, even with sample limited experiments. A platform based on high resolution nanoflow chromatography will be presented that uses the complementary advantages of both discovery and targeted proteomics. |
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The Essential Role of On-line Separations for Mass Spectrometry Based Proteomics Supported by Thermoelectron Corp
1:30 PM-4:20 PM, Sunday, 10 September 2006 Moscone Center -- Room 130, Oral
Division of Analytical Chemistry |