High-Throughput Proteomics and Phosphoproteomics of Rat Tissues Using Microflow Zeno SWATH

High-throughput tissue proteomics has great potential in the advancement of precision medicine. Here, we investigated the combined sensitivity of trap-elute microflow liquid chromatography with a ZenoTOF for DIA proteomics and phosphoproteomics. Method optimization was conducted on HEK293T cell lines to determine the optimal variable window size, MS2 accumulation time and gradient length. The ZenoTOF 7600 was then compared to the previous generation TripleTOF 6600 using eight rat organs, finding up to 23% more proteins using a fifth of the sample load and a third of the instrument time. Spectral reference libraries generated from Zeno SWATH data in FragPipe (MSFragger-DIA/DIA-NN) contained 4 times more fragment ions than the DIA-NN only library and quantified more proteins. Replicate single-shot phosphopeptide enrichments of 50–100 μg of rat tryptic peptide were analyzed by microflow HPLC using Zeno SWATH without fractionation. Using Spectronaut we quantified a shallow phosphoproteome containing 1000–3000 phosphoprecursors per organ. Promisingly, clear hierarchical clustering of organs was observed with high Pearson correlation coefficients >0.95 between replicate enrichments and median CV of 20%. The combined sensitivity of microflow HPLC with Zeno SWATH allows for the high-throughput quantitation of an extensive proteome and shallow phosphoproteome from small tissue samples.