Variable flow liquid chromatography-tandem mass spectrometry and the comprehensive analysis of complex protein digest mixtures
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A microscale, variable flow, liquid chromatography-electrospray ionization source has been coupled to an ion trap mass spectrometer and used to analyze both simple and complex protein mixtures. By using an analytical technique described as “peak parking,” the sample analysis times for components eluting from a liquid Chromatograph can be greatly increased without sacrificing Chromatographic resolution. This was achieved by instantaneously reducing the column head pressure (and hence, flowrate) under peaks of interest to prolong their elution from the tip. With the technique, it is possible to perform manual parent ion selection, higher resolution narrow mass range scans, and multiple stages of tandem mass spectrometry (MS2 and MS3) within the context of a single eluting peak. Benefits normally associated with an off-line tandem mass spectrometry analysis, such as the optimization of collision induced dissociation parameters and the analysis of more than one charge state, can now be obtained with the increased sensitivity and selectivity afforded by the chromatography. The utility of this method for the comprehensive analysis of complex mixtures was illustrated with the analysis of an in-gel protein digest mixture derived from a single spot from a two-dimensional electrophoresis gel. In addition to the expected enzyme digestion products, other peptides derived from nonspecific cleavage by the enzyme and protein impurities, as well as those having oxidized, derivatized, or deamidated amino acid residues were fully characterized.
KeywordsAutomatic Gain Control Methionine Sulfoxide Real Time Display Column Head Pressure Complex Peptide Mixture
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