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Mass spectrometry has established itself as the primary technique for identifying proteins due to its unparalleled speed, sensitivity, and specificity. Strategies for digestion of the proteins use a specific protease that cleaves at predictable residues along the peptide backbone, providing smaller stretches of peptide sequence more amenable to mass spectrometry analysis. When coupled with protein-level pre-fractionation strategies, such as one-dimensional PAGE, thus reducing the complexity of the protein mixture, this approach has proven highly successful in comprehensive protein identification and characterization. The downside of this approach is the number of gel samples, or fractions, to be analyzed by the LC-MS/MS system. With typical analytical HPLC run times of 45 min to 1 h, the amount of time required to analyze one top-level sample can be prohibitive.
Here we describe the use of elevated flow rates combined with nanoscale columns packed with sub-2-μm particles for rapid separations using a nanoUPLC system. Increasing the flow rate to 900 nL/min and running a very rapid gradient over 8 min on a 75 μm × 15 cm column, allows high-quality peptide separations to be achieved with a sample-to-sample inject time of 10 min. This, combined with an orthogonal acceleration time-of-flight mass spectrometer, using a newly developed high-speed data-dependent MS/ MS approach fragmenting up to eight precursor ions per second, allows for the rapid characterization of simple protein mixtures, such as those obtained from 1D gel bands.
We will present data from standard tryptic digests of known proteins and simple mixtures of protein digests used in the development of this method, and data from in-gel digests of 1D gel bands.