Orbitrap fusion mass spectrometer

BALF samples were sent to the Yale MS & Proteomics Resource (New Haven, CT), where they were processed and analyzed via a Label Free Quantification workflow as described by Charkoftaki et al. (25 (link)). The sample preparation was slightly modified from the 2019 protocol of Charkoftaki et al. (25 (link)) given the samples were BALF. In brief, samples were filtered through a 3-kDa Amicon Ultra filter, and the retentate was SpeedVac dried and used for downstream proteomics preparation. Dried protein pellets were reduced with DTT, alkylated with iodoacetamide, enzymatically digested with LysC and trypsin, and desalted using C18 RP microspin column. High-resolution liquid chromatography mass spectrometry (MS)/MS data were collected on an Orbitrap Fusion mass spectrometer coupled to a NanoACQUITY UPLC and analyzed using Progenesis QI (Waters, Milford, MA) and Mascot search engine. Quantitative data were normalized based on equal total amount of peptides/proteins injected on column, and positive protein identification and quantitation were based on hits with two or more unique peptides per protein. Experimental groups were compared and considered statistically significant when the FDR q value was <0.05, unless otherwise stated. IPA (Qiagen Bioinformatics) was used for further analyses.

Label-Free Quantitation (LFQ) was performed on a Thermo Scientific Orbitrap Fusion mass spectrometer connected to a Waters nano ACQUITY UPLC system equipped with a Waters Symmetry^® C18 180 μm × 20 mm trap column and a 1.7-μm, 75 μm × 250 mm nano ACQUITY UPLC column (maintained at 35°C). The digested protein samples were diluted to a final concentration of 0.05 μg protein/μL in 0.1% TFA. Five μL of these solutions were injected in triplicate into the LC MS/MS system in block randomized order. To ensure a high level of identification and quantitation integrity, a resolution of 120,000 and 60,000 was utilized for MS and MS/MS scans, respectively. Trapping was carried out for 3 min at 5 μL/min in 97% Buffer A (0.1% FA in water) and 3% Buffer B (0.075% FA in acetonitrile) prior to eluting with linear gradients that reached 6% B at 5 min, 35% B at 170 min, and 50% B at 175 min, and 97% B at 180 min for 5 min; then dropped down to 3% B at 186 min for 14 min.