Standardized sample preparation of paediatric bronchoalveolar lavage fluid for mass spectrometry based proteomic analysis

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BACKGROUND: Bronchoalveolar lavage fluid (BALF) is a valuable diagnostic and research tool in paediatric respiratory medicine. Mass spectrometry-based proteomic analysis of BALF can contribute to uncover disease mechanisms and biomarkers, but standardized protocols for paediatric BALF are lacking. This study aimed to establish a robust, reproducible workflow for liquid chromatography-tandem mass spectrometry (LC-MS/MS) of paediatric BALF and to evaluate its applicability in samples from patients with different clinical conditions. METHODS: BALF was collected from five children (ages 1–6 years) undergoing bronchoscopy for various indications. As a reference, we used an adult-derived workflow for BALF analysis, which combines ultracentrifugation (UC) and protein depletion. To address the lower protein yield in paediatric BALF and simplify the procedure, individual steps were systematically omitted, resulting in four workflows: UC plus depletion, UC only, depletion only, and a simplified workflow omitting both. All samples underwent 3 kDa ultrafiltration followed by protein digestion using the S-Trap methodology. Proteins were identified and quantified by LC-MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer. Reproducibility was assessed using technical replicates and BALF from all five patients was analysed to demonstrate applicability. Quantified proteins were further explored by Gene Ontology annotation and pathway mapping. RESULTS: Altogether, the four workflows quantified 635 proteins from digests standardized to 10 µg protein, readily obtained from as little as 1 ml BALF. Among individual workflows, the simplified workflow yielded the highest number of proteins, with 632 quantified in at least one patient. A core set of 425 proteins (75%) was consistently detected across all patients, regardless of diagnosis. The distribution of coefficient of variation across technical replicates was comparable between workflows. Notably, the simplified workflow reduced hands-on time by approximately five hours compared to the others. Many identified proteins were associated with salivary secretion, complement and coagulation cascades, and Intestinal immune network for IgA production. CONCLUSIONS: This study establishes an efficient and reproducible workflow for proteomic analysis of paediatric BALF requiring smaller sample volumes than typically available from adults. The simplified workflow achieved robust proteome coverage while minimizing sample loss, providing a practical basis for large-scale proteomic studies in paediatric respiratory diseases.

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