1. A Universal and High-Throughput Proteomics Sample Preparation Platform
- Author
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Christopher A. LeClair, Matthew D. Hall, Menghang Xia, Ruili Huang, Zhengxi Wei, Anton Simeonov, Tuan Xu, Qin Yao, Valeriu Cebotaru, Yuhong Fang, Andrew P. Burns, Yaqin Zhang, and Dingyin Tao
- Subjects
Proteomics ,Reproducibility ,Chromatography ,Proteome ,Chemistry ,010401 analytical chemistry ,Reproducibility of Results ,010402 general chemistry ,Mass spectrometry ,01 natural sciences ,High-performance liquid chromatography ,Sample (graphics) ,Article ,Mass Spectrometry ,0104 chemical sciences ,Analytical Chemistry ,Humans ,Sample preparation ,Target protein ,Chromatography, High Pressure Liquid - Abstract
Major advances have been made to improve the sensitivity of mass analyzers, spectral quality, and speed of data processing enabling more comprehensive proteome discovery and quantitation. While focus has recently begun shifting toward robust proteomics sample preparation efforts, a high-throughput proteomics sample preparation is still lacking. We report the development of a highly automated universal 384-well plate sample preparation platform with high reproducibility and adaptability for extraction of proteins from cells within a culture plate. Digestion efficiency was excellent in comparison to a commercial digest peptide standard with minimal sample loss while improving sample preparation throughput by 20- to 40-fold (the entire process from plated cells to clean peptides is complete in ~300 min). Analysis of six human cell types, including two primary cell samples, identified and quantified ~4000 proteins for each sample in a single high-performance liquid chromatography (HPLC)–tandem mass spectrometry injection with only 100 – 10,000 cells, thus demonstrating universality of the platform. The selected protein was further quantified using a developed HPLC-multiple reaction monitoring method for HeLa digests with a heavy labeled internal standard peptide spiked in. Excellent linearity was achieved across different cell numbers indicating a potential for target protein quantitation in clinical research.
- Published
- 2021