Spectral library search for improved TMTpro labelled peptide assignment in human plasma proteomics

Nicolai B. Palstrøm, Amanda J. Campbell, Caroline A. Lindegaard, Samir Cakar, Rune Matthiesen, Hans C. Beck*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review


Clinical biomarker discovery is often based on the analysis of human plasma samples. However, the high dynamic range and complexity of plasma pose significant challenges to mass spectrometry-based proteomics. Current methods for improving protein identifications require laborious pre-analytical sample preparation. In this study, we developed and evaluated a TMTpro-specific spectral library for improved protein identification in human plasma proteomics. The library was constructed by LC-MS/MS analysis of highly fractionated TMTpro-tagged human plasma, human cell lysates, and relevant arterial tissues. The library was curated using several quality filters to ensure reliable peptide identifications. Our results show that spectral library searching using the TMTpro spectral library improves the identification of proteins in plasma samples compared to conventional sequence database searching. Protein identifications made by the spectral library search engine demonstrated a high degree of complementarity with the sequence database search engine, indicating the feasibility of increasing the number of protein identifications without additional pre-analytical sample preparation. The TMTpro-specific spectral library provides a resource for future plasma proteomics research and optimization of search algorithms for greater accuracy and speed in protein identifications in human plasma proteomics, and is made publicly available to the research community via ProteomeXchange with identifier PXD042546.

Original languageEnglish
Publication statusE-pub ahead of print - 22. Jun 2023

Bibliographical note

Funding Information:
This work was supported by a research grant from the Danish Cardiovascular Academy, which is funded by the Novo Nordisk Foundation, grant number NNF20SA0067242 and the Danish Heart Foundation.

Publisher Copyright:
© 2023 The Authors. Proteomics published by Wiley-VCH GmbH.


  • peptide identification
  • plasma proteomics
  • spectral library search
  • TMTpro


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