NanoTPOT: Enhanced Sample Preparation for Quantitative Na-noproteomic Analysis (revision work)

With the ever-growing need for protein-level understanding in pathological research, proteomics researchers thrive to exam-ine 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 sen-sitive, quantitative and reproducible protein analysis for these increasingly sophisticated studies. Leveraging the benefits of an acid-cleavable detergent, RapiGest SF Surfactant (Waters Corporation), we developed and optimized a nanoproteomic workflow that we termed Nanogram TMT Processing in One Tube (NanoTPOT). Through the assessment of proteolytic di-gestion, tandem mass tag (TMT) labeling, online and offline fractionation strategies, our optimized workflow effectively eliminated the need for sample desalting and enabled compatible sample processing for mass spectrometry 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 and 5474 proteins in TMT 10-plex experiments with one microgram of lysate protein and 2000 sorted HeLa cells (500ng lysate protein) in each channel, respectively. Our workflow has been proven to be an effec-tive alternative for current nanoproteomic sample processing to minimize sample loss in biological and clinical applications.