Proteomics has typically been done using nanoflow LC for sensitivity but the time to results slow. With higher flow rates, sample can be loaded faster, trap/column can be washed and equilibrated faster, and gradients are formed faster, allowing much faster run times to be achieved. Microflow LC has been used increasing in quantitative proteomics in combination with SWATH® Acquisition, to provide better robustness and higher throughput when measuring larger sample cohorts. Here, the impact of gradient length on protein identification and quantitation results with DIA was explored.
Microflow LC was performed on the SCIEX TripleTOF® 6600 System with OptiFlow® Source using the nanoLC™ 425 system plumbed in microflow mode. Trap-elute workflow was used and a range of gradient lengths from 5 – 45 mins was explored, and key acquisition parameters for SWATH Acquisition were varied to optimize for the much faster run times. Data was processed with SWATH 2.0 microapp in PeakView® Software 2.2 and OneOmics™ in SCIEX Cloud.
Using complex digested cell lysates, SWATH Acquisition experiments were performed using gradient lengths ranging from 5-45 mins and protein quantitation results were assessed. Fast MS/MS acquisition rates were found to be critical because this enabled more, smaller variable Q1 windows to improve S/N for quantitation. Even with the fastest gradients, methods with 60-100 windows with very fast accumulation times of 15 msec improved results. Optimization results will be presented. Optimized methods were then used to compare quality of quantitation between long and shortened gradients and similar fold change values were measured confirming accelerated gradients can be used for industrialized quantitative proteomics.