Improving quantitation accuracy in isobaric-labeling mass spectrometry experiments with spectral library searching and feature-based peptide-spectrum match filter

Isobaric labeling relative quantitation is one of the dominating proteomic quantitation technologies. Traditional quantitation pipelines for isobaric-labeled mass spectrometry data are based on sequence database searching. In this study, we present a novel quantitation pipeline that integrates sequence database searching, spectral library searching, and a feature-based peptide-spectrum-match (PSM) filter using various spectral features for filtering. The combined database and spectral library searching results in larger quantitation coverage, and the filter removes PSMs with larger quantitation errors, retaining those with higher quantitation accuracy. Quantitation results show that the proposed pipeline can improve the overall quantitation accuracy at the PSM and protein levels. To our knowledge, this is the first study that utilizes spectral library searching to improve isobaric labeling-based quantitation. For users to conveniently perform the proposed pipeline, we have implemented the feature-based filter being executable on both Windows and Linux platforms; its executable files, user manual, and sample data sets are freely available at https://ms.iis.sinica.edu.tw/comics/Software_FPF.html. Furthermore, with the developed filter, the proposed pipeline is fully compatible with the Trans-Proteomic Pipeline.

Automated summary

In this study, a novel quantitation pipeline that combines sequence database searching, spectral library searching, and a feature-based peptide-spectrum-match (PSM) filter is proposed to improve the quantitation accuracy of isobaric labeling-based mass spectrometry. The results demonstrate that this pipeline can enhance the overall quantitation accuracy at both the PSM and protein levels. This is the first study to utilize spectral library searching to improve isobaric labeling-based quantitation.