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NanoTPOT: Enhanced Sample Preparation for Quantitative Nanoproteomic Analysis

ABSTRACT: In this study, we developed and optimized a nanoproteomic workflow that we termed Nanogram TMT Processing in One Tube (NanoTPOT). Through the assessment of proteolytic digestion, tandem mass tag (TMT) labeling, online and offline frac-tionation strategies, our optimized workflow effectively eliminated the need for sample desalting and enabled compatible sample processing for MS analysis. We further applied the NanoTPOT workflow to examine cellular response to stress caused by dithiothreitol in Hela cells, where we identified and quantified 6935 proteins in a TMT 10-plex experiment with one microgram of starting material in each channel.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Fibroblast, Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Yu Lu

LAB HEAD: Yu Lu

PROVIDER: PXD016960 | Pride | 2021-01-11

REPOSITORIES: Pride

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NanoTPOT: Enhanced Sample Preparation for Quantitative Nanoproteomic Analysis.

Wu Ruilin R Pai Akshat A Liu Lina L Xing Sansi S Lu Yu Y

Analytical chemistry 20200420 9

With the ever-growing need for protein-level understanding in pathological research, proteomics researchers thrive to examine detailed proteome dynamics using crucial, yet often limited, primary and clinical samples. Aside from mass spectrometer instrumentation advancement, a single-tube-based high-throughput sample processing workflow is imperative to ensure sensitive, quantitative, and reproducible protein analysis for these increasingly sophisticated studies. Leveraging the benefits of an aci ...[more]

PMID: 32255623

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