Cysteine-Directed Isobaric Labeling Combined with GeLC-FAIMS-MS for Quantitative Top-Down Proteomics

The quantification of proteoforms, i.e., all molecular
forms in
which proteins can be present, by top-down proteomics provides essential
insights into biological processes at the molecular level. Isobaric
labeling-based quantification strategies are suitable for multidimensional
separation strategies and allow for multiplexing of the samples. Here,
we investigated cysteine-directed isobaric labeling by iodoTMT in
combination with a gel- and gas-phase fractionation (GeLC-FAIMS-MS)
for in-depth quantitative proteoform analysis. We optimized the acquisition
workflow (i.e., the FAIMS compensation voltages, isolation windows,
acquisition strategy, and fragmentation method) using a two-proteome
mix to increase the number of quantified proteoforms and reduce ratio
compression. Additionally, we implemented a mass feature-based quantification
strategy in the widely used deconvolution algorithm FLASHDeconv, which
improves and facilitates data analysis. The optimized iodoTMT GeLC-FAIMS-MS
workflow was applied to quantitatively analyze the proteome of Escherichia coli grown under glucose or acetate as
the sole carbon source, resulting in the identification of 726 differentially
abundant proteoforms.