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, a data independent acquisition (DIA) approach, to provide better robustness and higher throughput when measuring larger sample cohorts.
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. 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.
Next, the newest approach to DIA, Scanning SWATH acquisition was explored to determine if further workflow improvements could be made. 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 across all.