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

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