Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Weise DO

PROVIDER: S-EPMC10068177 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Publications

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

Weise Danielle O DO Kruk Monica E ME Higgins LeeAnn L Markowski Todd W TW Jagtap Pratik D PD Mehta Subina S Mickelson Alan A Parker Laurie L LL Wendt Christine H CH Griffin Timothy J TJ

Clinical proteomics 20230402 1

<h4>Background</h4>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 ...[more]

PMID: 37005570

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Project description:BackgroundDifferentiating malignant lung tumors from benign pulmonary nodules is a great challenge. While the analysis of bronchoalveolar lavage fluid (BALF) is used for diagnosing infections and interstitial lung diseases, there is limited evidence to support its use for lung cancer diagnosis. This study aimed to interrogate the potential of using BALF cell-free DNA (cfDNA) to discriminate malignant lesions from benign nodules.MethodsFifty-three patients with solid pulmonary nodules (≤2 cm) were prospectively enrolled, including 21 confirmed with benign disease and 32 with malignant tumors. Mutations were profiled for 30 tumor tissues and 40 BALFs. Paired BALFs and plasma from 48 patients underwent DNA methylation profiling. A methylome-based classification model was developed for BALF and plasma separately.ResultsAmong the 30 patients with paired tissues and BALFs, 96.7% and 70% had alterations detected from their tissues (79 alterations) and BALFs (53 alterations), respectively. Using tissues as references, BALFs revealed 14 new alterations and missed 41. BALF mutation displayed a sensitivity of 71%, specificity of 77.8%, and accuracy of 72.5% in detecting lung cancer. BALF methylation achieved an accuracy of 81.3%, with both sensitivity and specificity being 81%. Plasma methylation showed a 66.7% sensitivity, 71.4% specificity, and 68.8% accuracy. BALF methylation also demonstrated 82.4% sensitivity in stage I patients. Parallel bronchoscopy, lavage cytology, and bronchial brushing demonstrated an inferior sensitivity of 23%, 3.1%, and 9.7%, respectively, compared with BALF methylation and mutation (P<0.0001).ConclusionsBALF cfDNA can serve as a liquid biopsy media for both mutation and methylation profiling, demonstrating better sensitivities in distinguishing small malignant tumors from benign nodules than conventional methods.KeywordsLung cancer diagnosis; pulmonary nodule; bronchoalveolar lavage fluid (BALF); methylation; genomic mutation.

Dataset Information

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

Background

Results

Conclusions

Publications

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

Similar Datasets

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