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Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole ‐ time‐of‐flight mass spectrometer

The application of microfabricated devices coupled to a quadrupole time‐of‐flight mass spectrometer (Qq‐TOF‐MS) is presented for the analysis of trace level digests of gel‐isolated proteins. In order to enhance the sample loading for proteomics analyses, two different on‐chip sample preconcentration techniques were evaluated. First, a sample stacking procedure that used polarity switching to remove the sample buffer prior to zone electrophoresis was easily integrated on the microfabricated devices. With the present chip design, this preconcentration technique provided up to 70 nL sample injection with sub‐nM detection limits for most peptide standards. For applications requiring larger sample loading, a disposable adsorption preconcentrator using a C18 membrane is incorporated outside the chip. This preconcentration method yielded lower peptide recoveries than that obtainable with sample stacking, and provided a convenient means of injecting several μL of sample with detection limits of typically 2.5 nM for hydrophobic peptides. The analytical merits of both sample enrichment approaches are described for the identification of bands isolated from two‐dimensional (2‐D) gel separation of protein extracts from Haemophilus influenzae. Accurate molecular mass measurements (< 5 ppm) in peptide mapping experiments is obtained by introducing an internal standard via a post‐separation channel. Rapid identification of trace level peptides is also demonstrated using on‐line tandem mass spectrometry and database searching with peptide sequence tags.