Dataset Information

Cysteine-directed isobaric labeling combined with GeLC-FAIMS MS for quantitative top-down proteomics

ABSTRACT: 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 multi-dimensional 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.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD055510 | Pride | 2025-05-07

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20240422_TM_Saccharomyces_Cervesiae_uniprot_canonical_and_isoforms.fasta	Fasta
	Ecoli_GvA_iodoTMTsix_PEPPI-40_2scan_peptideMode_highres_r1.raw	Raw
	Ecoli_GvA_iodoTMTsix_PEPPI-40_2scan_peptideMode_highres_r1_quant.tsv	Tabular
	Ecoli_GvA_iodoTMTsix_PEPPI-40_2scan_peptideMode_highres_r2.raw	Raw
	Ecoli_GvA_iodoTMTsix_PEPPI-40_2scan_peptideMode_highres_r2_quant.tsv	Tabular

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Publications

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

Matzanke Theo T Kaulich Philipp T PT Jeong Kyowon K Takemori Ayako A Takemori Nobuaki N Kohlbacher Oliver O Tholey Andreas A

Journal of proteome research 20250130 3

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 qu ...[more]

PMID: 39885717

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