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Comparative Analysis of Quantitative Mass Spectrometric Methods for Subcellular Proteomics
journal contribution
posted on 2020-03-05, 21:06 authored by Abla Tannous, Marielle Boonen, Haiyan Zheng, Caifeng Zhao, Colin J. Germain, Dirk F. Moore, David E. Sleat, Michel Jadot, Peter LobelKnowledge
of intracellular location can provide important insights
into the function of proteins and their respective organelles, and
there is interest in combining classical subcellular fractionation
with quantitative mass spectrometry to create global cellular maps.
To evaluate mass spectrometric approaches specifically for this application,
we analyzed rat liver differential centrifugation and Nycodenz density
gradient subcellular fractions by tandem mass tag (TMT) isobaric labeling
with reporter ion measurement at the MS2 and MS3 level and with two
different label-free peak integration approaches, MS1 and data independent
acquisition (DIA). TMT-MS2 provided the greatest proteome coverage,
but ratio compression from contaminating background ions resulted
in a narrower accurate dynamic range compared to TMT-MS3, MS1, and
DIA, which were similar. Using a protein clustering approach to evaluate
data quality by assignment of reference proteins to their correct
compartments, all methods performed well, with isobaric labeling approaches
providing the highest quality localization. Finally, TMT-MS2 gave
the lowest percentage of missing quantifiable data when analyzing
orthogonal fractionation methods containing overlapping proteomes.
In summary, despite inaccuracies resulting from ratio compression,
data obtained by TMT-MS2 assigned protein localization as well as
other methods but achieved the highest proteome coverage with the
lowest proportion of missing values.
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label-free peak integration approachesratio compressionMS 3 levelNycodenz density gradient subcellular fractionsTMT-MS 2Subcellular Proteomics KnowledgeQuantitative Mass Spectrometric Methodsproteome coveragetandem mass tagisobaricDIAproteinreporter ion measurementorthogonal fractionation methodsdatalocalization
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