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Two-dimensional separation by nano-LC and trapped ion mobility spectrometry (TIMS) prior to Q/TOF tandem mass spectrometry significantly improves the accuracy of isobaric tag-based quantitation in proteome analysis without the need for additional measurement time for TIMS insertion between LC and Q/TOF MS. The obtained peak capacity of up to 3300 h -1 in LC/TIMS reduced the coisolation of precursor ions at the quadrupole analyzer, resulting in more accurate ratios of reporter ions derived from isobaric tags in product ion spectra obtained at the TOF analyzer. We also found that TIMS with a narrower quadrupole isolation window could reduce the ratio compression effect at least as effectively as the synchronous precursor selection method using MS3 scans without compromising sensitivity or coverage. Our results suggest that the 65 min gradient LC/TIMS/Q/TOF system is an excellent platform for high-throughput proteomics studies.

Extending the Separation Space with Trapped Ion Mobility Spectrometry Improves the Accuracy of Isobaric Tag-Based Quantitation in Proteomic LC/MS/MS