We report a data independent acquisition (DIA) strategy that dynamically adjusts the tandem mass spectrometry (MS/MS) windows during the chromatographic separation. The method focuses MS/MS acquisition on the most relevant mass range at each point in time – improving the quantitative sensitivity by increasing the time spent on each DIA window. We demonstrate an improved lower limit of quantification, on average, without sacrificing the number of peptides detected.

Quantitative comparison of traditional DIA methods with a novel dynamic DIA approach.

HeLa cell lysates were prepared using an automated single pot solid phase sample preparation (SP3) protocol.14 Briefly, cell pellets were lysed in 2% SDS with 10 seconds of probe sonication. Total protein concentration was estimated using a Pierce BCA assay (Thermo Fisher Scientific) and 1% SDS solution was added to bring the final concentration to approximately 4 μg/μL. Proteins were reduced in 20 mM dithiothreitol and alkylated in 40 mM iodoacetamide. Following reduction and alkylation, each sample was diluted to 70% acetonitrile and bound to MagResyn Hydroxyl particles (Resyn Biosciences) at a ratio of 100:1 (beads to protein) for a total of 10 minutes. Subsequent washing and digestion steps were performed on a Kingfisher Flex (Thermo Fisher Scientific). The samples were washed three times in 95% acetonitrile and twice in ethanol. Then, samples were digested with trypsin in 50 mM ammonium bicarbonate at an enzyme to protein ratio of 1:25. The peptide samples were dried down via vacuum centrifugation and resuspended in 0.1% formic acid. A dilution curve was made by diluting the normal digest in the SILAC labeled digest with an equal amount of Pierce Peptide Retention Time Calibration standard (Thermo Fisher Scientific) spiked into each sample. A total of 11 dilution points were made, consisting of the following fractions of normal digest: 0, 0.5, 1, 3, 5, 7, 10, 30, 50, 70, and 100%.