RESUMEN
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.
RESUMEN
Chronic obstructive pulmonary disease (COPD) is among the leading causes of death worldwide and HIV is an independent risk factor for the development of COPD. However, the etiology of this increased risk and means to identify persons with HIV (PWH) at highest risk for COPD have remained elusive. Biomarkers may reveal etiologic pathways and allow better COPD risk stratification. We performed a matched case:control study of PWH in the Strategic Timing of Antiretoviral Treatment (START) pulmonary substudy. Cases had rapid lung function decline (> 40 mL/year FEV1 decline) and controls had stable lung function (+ 20 to - 20 mL/year). The analysis was performed in two distinct groups: (1) those who were virally suppressed for at least 6 months and (2) those with untreated HIV (from the START deferred treatment arm). We used linear mixed effects models to test the relationship between case:control status and blood concentrations of pneumoproteins (surfactant protein-D and club cell secretory protein), and biomarkers of inflammation (IL-6 and hsCRP) and coagulation (d-dimer and fibrinogen); concentrations were measured within ± 6 months of first included spirometry. We included an interaction with treatment group (untreated HIV vs viral suppression) to test if associations varied by treatment group. This analysis included 77 matched case:control pairs in the virally suppressed batch, and 42 matched case:control pairs in the untreated HIV batch (n = 238 total) who were followed for a median of 3 years. Median (IQR) CD4 + count was lowest in the controls with untreated HIV at 674 (580, 838). We found no significant associations between case:control status and pneumoprotein or biomarker concentrations in either virally suppressed or untreated PWH. In this cohort of relatively young, recently diagnosed PWH, concentrations of pneumoproteins and biomarkers of inflammation and coagulation were not associated with subsequent rapid lung function decline.Trial registration: NCT00867048 and NCT01797367.