Sample Processing Protocol: BALF samples were thawed and centrifuged. The supernatant was removed, frozen, and lyophilized. Samples were resuspended in LC-MS grade water and ultra-centrifuged at 100,000 x G. The supernatant was transferred to a 4 mL 3 kDa MWCO filter and ultra-centrifuged to concentrate the sample and simultaneously collect endogenous peptides in the flow-through. The concentrated protein was quantified and loaded into Seppro IgY14 spin columns to deplete 14 highly abundant plasma proteins. The BALF flow-through, collected from Seppro, was transferred to a 0.5 mL 3 kDa MWCO filter and ultra-centrifuged to concentrate the sample. The concentrated protein was quantified, frozen, and lyophilized. Dried BALF samples were solubilized in 5% SDS, sonicated, and centrifuged. The supernatant was reduced, alkylated, and acidified to pH < 1. Samples were transferred to S-trap columns where the protein was trapped, washed to remove contaminants, and digested overnight with Trypsin Gold, MS grade. Digested proteins were eluted from the column, frozen, and lyophilized. BALF peptides were quantified via Nanodrop and analyzed via LC-MS/MS. 300 nanograms of each peptide fraction was analyzed on-line with an Orbitrap Eclipse mass spectrometer with FAIMS separation. Additional sample processing after peptide quantification included TMT labeling and fractionation of BALF protein digest. BALF peptides were labeled with TMT16pro reagents, pooled, frozen, and lyophilized. Pooled, labeled, BALF samples were fractionated with offline high pH fractionation or microscale fractionation. The TMT-labeled peptide mixtures were analyzed via LC-MS/MS – 800 nanograms of each fraction was analyzed on-line with an Orbitrap Eclipse mass spectrometer with FAIMS separation. Data Processing Protocol: Sequence database searching was performed by processing peptide tandem MS using SEQUEST in Proteome Discoverer 2.5. The human Universal Proteome protein sequence database was merged with a common lab contaminant protein database. The search parameters were: enzyme trypsin full specificity, 2 missed cleave sites; peptide length 6 – 50 amino acids, precursor tolerance 15 ppm, and fragment ion tolerance was 0.06 DA. CAM cysteine was specified as a fixed modification. A 1% protein and peptide False Discovery Rate was applied. Proteome Discoverer 2.5 was also used for TMT-based protein quantification

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. Note: Due to the nature of some of the files, file wendt005_ostr0103_18260_20210831_BALF_FAIMS_MS2_TMT16.msf, wendt005_ostr0103_18976_20230202_quantReport.msf, cmsptc_higgi022_18988_20230203_18976DW_EnF_hcdlT_1R.raw, cmsptc_higgi022_18988_20230203_18976DW_EnF_hcdlT_2R.raw, cmsptc_higgi022_18988_20230203_18976DW_EnF_hcdlT_3R.raw and cmsptc_higgi022_18988_20230203_18976DW_Eclipse_noFAIMS_quantReport.msf were zipped into compressed folders before uploading.