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Proteomic analysis of primary graft dysfunction and high particle flow rate

ABSTRACT: Background: Primary graft dysfunction (PGD) remains a challenge to lung transplantation (LTx) recipients as a leading cause of poor early outcomes. New methods are needed for the rapid detection of PGD and the measurement of particle flow rate (PFR) from exhaled breath is a novel means to monitor disease. Methods: 22 recipient pigs underwent orthotopic left LTx and were evaluated for PGD on the third post-operative day. Exhaled breath particles (EBPs) and PFR were measured on mechanical ventilation. EBPs were evaluated with mass spectrometry and the proteome was compared to tissue biopsies and bronchoalveolar lavage fluid (BALF). Findings were confirmed in EBPs from 11 human transplant recipients. Results: 9 recipients developed PGD and had significantly higher PFR (686.4 (449.7-8824.0) particles per minute (ppm)) compared to recipients without PGD (116.6 (79.7-307.4) ppm, p=0.0005). From proteomic analysis, porcine and human EBP proteins recapitulated the BAL and adherens and tight junction proteins were underexpressed in PGD tissue. Conclusions: Histological and proteomic analysis found significant changes to the alveolar-capillary barrier to explain the increased PFR in recipients with PGD. Combined with the similarity of proteomic profiles between EBPs and BALF, exhaled breath measurement is proposed as a rapid and non-invasive bedside measurement of PGD.

INSTRUMENT(S): timsTOF Pro 2, timsTOF SCP

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Lung, Bronchoalveolar Lavage

SUBMITTER: Gabriel Hirdman

LAB HEAD: Sandra Lindstedt

PROVIDER: PXD046365 | Pride | 2024-04-05

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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Proteomic analysis of primary graft dysfunction and high particle flow rate

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