An optimized workflow for MS-based quantitative proteomics of challenging clinical bronchoalveolar lavage fluid (BALF) samples

Research output: Contribution to journalArticlepeer-review


Background: Clinical bronchoalveolar lavage fluid (BALF) samples are rich in biomolecules, including proteins, and useful for molecular studies of lung health and disease. However, mass spectrometry (MS)-based proteomic analysis of BALF is challenged by the dynamic range of protein abundance, and potential for interfering contaminants. A robust, MS-based proteomics compatible sample preparation workflow for BALF samples, including those of small and large volume, would be useful for many researchers. Results: We have developed a workflow that combines high abundance protein depletion, protein trapping, clean-up, and in-situ tryptic digestion, that is compatible with either qualitative or quantitative MS-based proteomic analysis. The workflow includes a value-added collection of endogenous peptides for peptidomic analysis of BALF samples, if desired, as well as amenability to offline semi-preparative or microscale fractionation of complex peptide mixtures prior to LC–MS/MS analysis, for increased depth of analysis. We demonstrate the effectiveness of this workflow on BALF samples collected from COPD patients, including for smaller sample volumes of 1–5 mL that are commonly available from the clinic. We also demonstrate the repeatability of the workflow as an indicator of its utility for quantitative proteomic studies. Conclusions: Overall, our described workflow consistently provided high quality proteins and tryptic peptides for MS analysis. It should enable researchers to apply MS-based proteomics to a wide-variety of studies focused on BALF clinical specimens.

Original languageEnglish (US)
Article number14
JournalClinical Proteomics
Issue number1
StatePublished - Dec 2023

Bibliographical note

Funding Information:
We thank the Center for Metabolomics and Proteomics at the University of Minnesota for providing services related to MS-based proteomics analysis, including consulting on sample preparation methods, generating MS-based data and analysis of results. The views expressed in this article are those of the authors and do not reflect the views of the United States Government, the Department of Veterans Affairs, the funders, the sponsors, or any of the authors’ affiliated academic institutions.

Funding Information:
This work was supported by Grant R01HL140971 from the NIH to C.H.W and T.J.G. The Orbitrap Eclipse instrumentation platform used in this work was purchased through High-end Instrumentation Grant S10OD028717 from the NIH.

Publisher Copyright:
© 2023, The Author(s).


  • BALF
  • Bronchoalveolar lavage fluid
  • Lung disease
  • Mass spectrometry
  • Quantitative proteomics
  • Sample preparation

PubMed: MeSH publication types

  • Journal Article


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