One-STAGE Tip Method for TMT-Based Proteomic Analysis of a Minimal Amount of Cells

Liquid chromatography-tandem mass spectrometry (LC-MS)-based profiling of proteomes with isobaric tag labeling from low-quantity biological and clinical samples, including needle-core biopsies and laser capture microdissection, has been challenging due to the limited amount and sample loss during preparation. To address this problem, we developed OnM (On-Column from Myers et al. and mPOP)-modified on-column method combining freeze-thaw lysis of mPOP with isobaric tag labeling of On-Column method to minimize sample loss. OnM is a method that processes the sample in one-STAGE tip from cell lysis to tandem mass tag (TMT) labeling without any transfer of the sample. In terms of protein coverage, cellular components, and TMT labeling efficiency, the modified On-Column (or OnM) displayed similar performance to the results from Myers et al. To evaluate the lower-limit processing capability of OnM, we utilized OnM for multiplexing and were able to quantify 301 proteins in a TMT 9-plex with 50 cells per channel. We optimized the method as low as 5 cells per channel in which we identified 51 quantifiable proteins. OnM method is a low-input proteomics method widely applicable and capable of identifying and quantifying proteomes from limited samples, with tools that are readily available in a majority of proteomic laboratories.