Deep intact proteoform quantification of low-microgram-scale proteome by tandem mass tag labeling and online 2D high-pH/low-pH RPLC top-down analysis.

Isobaric chemical tag labels are the 'gold standard' for quantifying proteins in bottom-up proteomics. Recently, our group developed and optimized an intact protein-level tandem mass tag (TMT) labeling platform to identify and quantify intact proteoforms in complex biological samples. This intact-protein TMT labeling strategy enables multiplexed quantification and minimizes variability introduced during downstream sample preparation. Here, to achieve deeper proteome coverage, we developed an integrated TMT-labeling and online 2D high-pH/low-pH RPLC top-down workflow for proteoform quantification in complex cell lysates. TMT-labeled intact proteoforms derived from HeLa lysate were mixed at defined ratios and analyzed using the developed platform. The measured TMT reporter ion ratios for detected intact proteoforms closely matched the expected theoretical values, demonstrating high quantitative accuracy. The platform was further applied to quantify changes in proteoform abundance induced by staurosporine (STS) in HeLa cells. Using six channels of TMT10plex reagents, with 3.3 μg of protein labeled per channel and 20 μg total protein injected, we identified 47 proteoforms from 21 proteins exhibiting significant abundance changes in microgram-level samples. Functional enrichment analysis using DAVID associated these proteoforms with metabolic and apoptotic pathways previously linked to STS-induced cellular responses. These results underscore the platform's ability to resolve biologically relevant proteoform-level regulation. This work establishes a multiplexed, quantitative, multidimensional top-down proteomics platform that enables deep proteoform characterization using only microgram-level sample amounts. Data are available via ProteomeXchange with identifier PXD078120.