NanoTPOT: Enhanced Sample Preparation for Quantitative Nanoproteomic Analysis

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.