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Data for manuscript, “An optimized workflow for MS-based quantitative proteomics of challenging clinical bronchoalveolar lavage fluid (BALF) samples”

ABSTRACT: Clinical BALF samples are rich in biomolecules, including proteins, and useful for molecular studies of lung health and disease. However, MS based proteomic analysis of BALF is impeded by the dynamic range of protein abundance, and potential for interfering contaminants. We have developed a workflow that eliminates these challenges. By combining high abundance protein depletion, protein trapping, clean-up, and in-situ tryptic digestion, our workflow is compatible with both qualitative and quantitative MS-based proteomic analysis. The workflow includes collection of endogenous peptides for peptidomic analysis of BALF, if desired, as well as amenability to offline semi-preparative or microscale fractionation of peptide mixtures prior to LC-MS/MS analysis, for increased depth of analysis. We show the effectiveness of this workflow on BALF samples from COPD patients. Overall, our workflow should allow MS-based proteomics to be applied to a wide variety of studies focused on BALF clinical samples.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung

DISEASE(S): Chronic Obstructive Pulmonary Disease

SUBMITTER: Danielle Weise

LAB HEAD: Timothy J. Griffin

PROVIDER: PXD038522 | Pride | 2023-04-05

REPOSITORIES: Pride

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Project description:The goal of this study was to investigate the effects of magnetic iron cobalt oxide nanoparticles (cobalt doped Fe3O4 nanoparticles with increasing amounts of cobalt) after pulmonary exposure while in parallel presenting a proteomics platform that is easily transferable to large-scale nanotoxicology screening as part of an integrated assessment and testing approach for regulation of nanoparticles. Bronchoalveolar lavage fluid (BALF) is a proximal biofluid that can be used to monitor airway inflammation and toxic responses in the lung. It is routinely sampled for differential lung diagnostics and has been discussed as a source for early detection of lung cancer. In order to assess effects of metal oxide nanoparticles upon inhalation, bronchoalveolar lavage fluid from mice dosed by single intratracheal instillation was collected and subjected to classical biocompatibility assays as well as proteome analysis. Magnetic oxide nanoparticles with iron and cobalt oxide (Fe3-xCoxO4) at different ratios (1:0, 3:1, 1:1, 1:3, 0:1) were tested at two concentrations (54 µg, 162 µg per animal) and two time points after instillation (day 1, day 3). As a positive control carbon black nanomaterial known to induce lung inflammation was included for both time points, but only at the high dosage (162 µg per animal). Proteomics experiments were divided into three parts – test of reproducibility, discovery and screening phase. The reproducibility of the newly introduced Evosep One LC system was evaluated by re-measuring of technical replicates (n=16). During the discovery phase, selected representative samples with 3 biological replicates per group (total n=9) including pure iron oxide nanoparticles, pure cobalt oxide nanoparticles and vehicle controls were subjected to in-depth proteome profiling by extensive pre-fractionation and including isobaric tandem mass tag (TMT) labeling following a classical LC-MS/MS setup. This step allowed us to identify affected pathways and generate hypotheses regarding mechanisms of the effects of nanoparticles. During screening, all samples of the study were measured label-free as single-shot injections separated on short gradients of 21 min using the robust, high-throughput Evosep One LC system. This step allowed a fast screening of the 5 different types of magnetic metal oxide nanoparticles on BALF, at two concentrations and two time points together with their representative controls (total n=166). All screening measurements were completed in only 2.7 days.

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Data for manuscript, “An optimized workflow for MS-based quantitative proteomics of challenging clinical bronchoalveolar lavage fluid (BALF) samples”

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