Chronic obstructive pulmonary disease (COPD) is the second leading cause of death in India, primarily attributed to smoking. Asymptomatic smokers can develop COPD due to a combination of genetic, environmental, and molecular factors, highlighting the importance of early detection. Data-independent acquisition mass spectrometry (DIA-MS) proteomics provides an unbiased approach to analyzing proteomic profiles. This study is the first to utilize DIA-based proteomics to examine individual serum samples from three distinct male groups: healthy individuals (n=10), asymptomatic smokers (n=10), and COPD patients (n=10). This thorough analysis identified 667 proteins with a 1% false discovery rate. Differential expression analysis revealed 40 proteins distinguishing normal from asymptomatic smokers, 88 proteins differentiating COPD from healthy individuals, and 40 proteins differentiating COPD from asymptomatic smokers. Notably, proteins such as PRDX6, ELANE, PRKCSH, PRTN3, and MNDA may help distinguish COPD from asymptomatic smokers, while ELANE, H3-3A, IGHE, SLC4A1, and SERPINA11 could differentiate COPD from healthy subjects. Pathway enrichment and protein-protein interaction analyses highlighted significant changes in hemostasis, immune system functions, fibrin clot formation, and post-translational protein modifications. Key proteins were validated through a parallel reaction monitoring assay. Our findings identify critical protein markers in COPD patients, asymptomatic smokers, and healthy individuals, providing insights for clinicians to better understand the disease’s pathobiology, enhance disease management, and improve patient quality of life

The proteins identified in the proteomics data were further validated using a PRM assay. Skyline (64-bit) version 23.1.0.455 was employed to create the isolation list for protein-associated genes. The missed cleavage parameter was set to 0. For the transition parameters, the precursor charge was set to +2, while the product charges were set to +1 and +2, with y and p specified as the ion types. PRM data acquisition was performed using the Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific), integrated with the EASY-nLC 1200 system. One microgram of peptides was loaded onto a 50 cm analytical column (PepMap™ RSLC C18, 2 µm, 100 Å, 75 µm x 50 cm) and eluted at a flow rate of 300 nL/min using a binary buffer system consisting of 0.1% FA MS water as buffer A and 80% ACN 0.1% FA MS water as buffer B. The data acquired were analyzed with Skyline software, and the PRM spectral data were matched against the built-in spectral libraries within Skyline.

About 15 µL of serum was added to Pierce Top 14 Abundant Protein Depletion Spin Columns (Thermo Fisher Scientific) and incubated for 1 hour on a rotating shaker. After incubation, the depleted serum samples were eluted by centrifugation at 1500 g for 2 minutes at room temperature. The eluent was then concentrated to a quarter of its original volume using a SpeedVac. To measure protein concentration, the Bradford assay was conducted on the depleted serum samples at a 1:20 dilution. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) was used to visualize differential protein patterns. For in-solution digestion, 30 µg of protein from the depleted serum was utilized. The sample volume was first adjusted to 20 µL by adding 6 M urea, followed by the addition of 20 mM tris(2-carboxyethyl)phosphine (TCEP) and incubation at 37 °C for 1 hour. Next, 40 mM IAA was added, and the mixture was incubated in the dark at room temperature for 15 minutes. The pH was adjusted to 8 using 50 mM ammonium bicarbonate (ABC), added in increments until the desired pH was achieved. Trypsin digestion was carried out by adding 4 µL of trypsin (0.25 µg/µL) at a 1:30 ratio and incubating overnight at 37°C. The following day, the mixture was completely dried using a SpeedVac. The dried peptides were reconstituted in 0.1% trifluoroacetic acid (TFA) and purified using SepPak® C18 cartridges. The purification process included conditioning with a 10:90 MS water solution, equilibration with 0.1% TFA, washing with 0.1% TFA, desalting with a 5:95 MS water solution, and elution with 50% acetonitrile (ACN). The cleaned samples were completely dried again using a SpeedVac and dissolved in 15 µL of 2% ACN 0.1% formic acid (FA). Peptide quantification was performed at 205 and 280 nm before the samples were submitted for mass spectrometry analysis.