Comparison of DIA and TMT based protein quantification in complex background

ABSTRACT: Label free quantification (LFQ) and isobaric labelling quantification (ILQ) are among the most popular protein quantification workflows in discovery proteomics. Here, we compared the TMT 10-plex workflow to label free single shot data-independent acquisition (DIA) method on a controlled sample set. The sample set consisted of ten samples derived from 10 different mouse cerebelli spiked with the UPS2 protein standard in five different concentrations. To match instrument time between the methods, the combined TMT sample was fractionated into ten fractions. The LC-MS data were acquired at two facilities to assess inter-laboratory reproducibility. Both methods resulted in a high proteome coverage (>5,000 proteins) with low missing values on protein level (<2%) The TMT workflow led to 15-20% more identified proteins and a slightly better quantitative precision whereas the quantitative accuracy was better for the DIA method. The quantitative performance was benchmarked by the number of true positives (UPS2 proteins) within the top 100 candidates. TMT and DIA performed similar. The quantitative performance of the DIA data could be even improved by searching them directly against a database instead of using a project specific library. Our experiments also demonstrated that both methods can be easily transferred between facilities.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Jan Muntel  

LAB HEAD: Lukas Reiter

PROVIDER: PXD011691 | Pride | 2019-01-24

REPOSITORIES: Pride