As the field of quantitative proteomics continues to evolve, larger biological cohorts are being studied, often using precious samples obtained from biobanks or other difficult-to-obtain sources. This creates 2 workflow requirements: the need to acquire quantitative data on the digested samples faster and the need to use smaller amounts of sample. For these types of studies, data-independent acquisition (DIA) continues to grow as the workflow of choice for reproducible quantitative analysis of large numbers of proteins from a proteomic sample.
As such, new workflows and software tools have emerged. Previously, the combination of fast microflow chromatography and SWATH DIA enabled large numbers of proteins to be quantified from complex proteomics samples at very high rates, up to 100 samples per day.1 Zeno MS/MS on the ZenoTOF 7600 system provides ~5- to 6-fold increase in peptide MS/MS sensitivity and can be used in MRMHR, DDA and DIA workflows.2,3,4 Also, multiple powerful algorithms have emerged that have enabled more proteins to be identified and quantified from DIA data, such as DIA-NN software.
Here, the improvements in proteins identified and quantified using microflow SWATH DIA coupled with Zeno MS/MS is described. Four different gradient lengths (5, 10, 20 and 45 minutes) were tested to cover a range of application needs. The library-free approach to processing DIA data (using in silico generated spectral libraries) was also evaluated vs. the traditional shotgun proteomics approach with Zeno DDA. Other workflow comparisons were performed to benchmark the workflows. DIA data were processed with DIA-NN software and DDA data were processed with ProteinPilot app in OneOmics suite.