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

Abstract

Liquid chromatography-tandem mass spectrometry (LC-MS)-basedprofiling of proteomes with isobaric tag labeling from low-quantitybiological and clinical samples, including needle-core biopsies andlaser capture microdissection, has been challenging due to the limitedamount and sample loss during preparation. To address this problem,we developed OnM (On-Column from Myers et al. and mPOP)-modified on-columnmethod combining freeze-thaw lysis of mPOP with isobaric taglabeling of On-Column method to minimize sample loss. OnM is a methodthat processes the sample in one-STAGE tip from cell lysis to tandemmass tag (TMT) labeling without any transfer of the sample. In termsof protein coverage, cellular components, and TMT labeling efficiency,the modified On-Column (or OnM) displayed similar performance to theresults from Myers et al. To evaluate the lower-limit processing capabilityof OnM, we utilized OnM for multiplexing and were able to quantify301 proteins in a TMT 9-plex with 50 cells per channel. We optimizedthe method as low as 5 cells per channel in which we identified 51quantifiable proteins. OnM method is a low-input proteomics methodwidely applicable and capable of identifying and quantifying proteomesfrom limited samples, with tools that are readily available in a majorityof proteomic laboratories.