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High-Throughput Proteomics and Phosphoproteomics of Rat Tissues Using Microflow Zeno SWATH
Erin M Humphries1, Dylan Xavier1, Keith Ashman2
1ProCan®, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, New South Wales 2145, Australia.
Journal of Proteome Research
|May 31, 2024
Summary
This study enhances precision medicine by combining microflow liquid chromatography with ZenoTOF for high-throughput proteomics and phosphoproteomics. The new method significantly increases protein identification from smaller tissue samples with reduced instrument time.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- High-throughput tissue proteomics is crucial for advancing precision medicine.
- Current methods face limitations in sensitivity and sample input requirements.
Purpose of the Study:
- To investigate the combined sensitivity of microflow liquid chromatography and ZenoTOF for DIA (Data-Independent Acquisition) proteomics and phosphoproteomics.
- To optimize methods for enhanced protein and phosphoprotein quantitation from small tissue samples.
Main Methods:
- Method optimization using HEK293T cell lines (variable window size, MS2 accumulation time, gradient length).
- Comparison of ZenoTOF 7600 with TripleTOF 6600 using eight rat organs.
- Generation of spectral reference libraries using FragPipe (MSFragger-DIA/DIA-NN).
- Analysis of phosphopeptide enrichments using microflow HPLC and Zeno SWATH.
Main Results:
- The ZenoTOF 7600 identified up to 23% more proteins with significantly less sample and instrument time compared to the TripleTOF 6600.
- Zeno SWATH data libraries contained 4x more fragment ions, leading to increased protein quantification.
- Quantified 1000-3000 phosphoprecursors per organ, demonstrating a shallow phosphoproteome.
- High reproducibility observed with Pearson correlation coefficients >0.95 and median CV of 20%.
Conclusions:
- The combined sensitivity of microflow HPLC with Zeno SWATH enables high-throughput quantitation of extensive proteomes and phosphoproteomes from limited tissue samples.
- This approach holds significant potential for precision medicine applications requiring detailed molecular profiling.
- The optimized workflow provides a powerful tool for deep proteomic and phosphoproteomic analysis.
