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.