The use of microflow LC in combination with SWATH® Acquisition has been growing steadily for quantitative proteomics as it provides better robustness and higher throughput when measuring larger sample cohorts. While variable window SWATH provides very good quantitative results on faster gradients, more narrow peak widths require faster cycle times and therefore present an opportunity for further acquisition improvements. With Scanning SWATH acquisition, a narrow Q1 window can be very rapidly scanned across a Q1 mass range and MS/MS is acquired at every small step. This adds an extra dimension to the DIA data that can be used to improve data quality.
Microflow LC was performed on the SCIEX TripleTOF® 6600 System with the OptiFlow® Source using the nanoLC™ 425 system. A trap-elute workflow was used and a range of gradient lengths from 5 – 45 mins was explored, with key acquisition parameters for Scanning SWATH Acquisition varied to optimize and compare to variable window SWATH acquisition using a research prototype Analyst® Software. Data was processed with research prototype SWATH software and results were analyzed using the SWATH Replicates Template.
Using a variety of complex matrices, both single species and mixtures of digests, acquisition parameters for Scanning SWATH Acquisition was explored. Not surprisingly, using a more narrow Q1 window provided improvements in # of peptides quantified over variable window SWATH for the microflow gradients tested. Optimization results will be presented.