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

Comprehensive global proteome profiling that is amenable
to high
throughput processing will broaden our understanding of complex biological
systems. Here, we evaluate 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
marginally higher 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.