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Isobaric Tagging and Data Independent Acquisition as Complementary Strategies for Proteome Profiling on an Orbitrap Astral Mass Spectrometry

ABSTRACT: Comprehensive global proteome profiling that is amenable to high throughput processing will broaden our understanding of complex biological systems. Here, we evaluated two leading mass spectrometry techniques—Data-Independent Acquisition (DIA) and Tandem Mass Tagging (TMT)—for extensive protein abundance profiling. DIA provides label-free quantification with a broad dynamic range, while TMT enables multiplexed analysis using isobaric tags for efficient cross-sample comparisons. We analyzed 18 samples, including four cell lines (IHCF, HCT116, HeLa, MCF7) under standard growth conditions, in addition to IHCF treated with two H₂O₂ concentrations, all in triplicate. Experiments were conducted on an Orbitrap Astral mass spectrometer, employing Field Asymmetric Ion Mobility Spectrometry (FAIMS). Despite utilizing different acquisition strategies, both the DIA and TMT approaches achieved comparable proteome depth and quantitative consistency, with each method quantifying over 10,000 proteins across all samples, with slightly more protein-level precision for the TMT strategy. Relative abundance correlation analysis showed strong agreement at both peptide and protein levels. Our findings highlight the complementary strengths of DIA and TMT for high-coverage proteomic studies, providing flexibility in method selection based on specific experimental needs.

INSTRUMENT(S): Orbitrap Astral

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Joao Paulo

LAB HEAD: Joao A. Paulo

PROVIDER: PXD058918 | Pride | 2025-02-14

REPOSITORIES: pride

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	az03970.raw	Raw
	az03970_XL_As_AB24_fin.mzIdentML	Mzid

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