Data independent acquisition (DIA) strategies have been used to increase the comprehensiveness of data collection while maintaining high quantitative reproducibility. In DIA, larger fixed-width Q1 windows are stepped across the mass range in an LC timescale, transmitting populations of peptides for fragmentation, and high resolution MS and MS/MS spectra are acquired. Previous work has shown that using more narrow variable width Q1 windows can improve peptide detection and increase sample coverage. Many labs are now using DIA to perform larger scale quantitative proteomic experiments with solid reproducibility on 1000s of proteins in complex matrices. As this technique increasingly proves to be a solid tool for biomarker research, larger sample sets are being analyzed, driving the need for further investigation of workflow improvements for throughput and robustness.

Here microflow LC was investigated in combination with SWATH^® acquisition on a number of complex matrices, to assess depth of coverage and robustness relative to current nanoflow strategies. The MS analysis was performed on a TripleTOF^® 6600 System (SCIEX) equipped with an nanoLC™ 425 system (SCIEX) with microflow modules. A number of acquisition parameters were explored to understand the workflow options in this flow regime. Key workflow recommendations have been established to provide researchers additional strategies for performing large scale, higher throughput SWATH acquisition studies.