In-silico immune cell deconvolution of the airway proteomes of infants with pneumonia reveals a link between reduced airway eosinophils and an increased risk of mortality

Charles J Sande; Jacqueline M Waeni; James M Njunge; Martin N Mutunga; Elijah Gicheru; Nelson K Kibinge; Agnes Gwela

The samples were centrifuged at 8,000xg for 10 minutes at 4°C to obtain the protein pellets and supernatants were discarded. The protein pellet was resuspended in 100µl of 50mM Triethylammonium bicarbonate (TEAB, Sigma-Aldrich, USA). Trypsin (Sigma-Aldrich, USA) was added to the protein samples at a trypsin-protein sample ratio of 1:10 and protein digestion was allowed to proceed overnight at 37°C with shaking. The peptide samples were randomly assigned to 10 individual batches: each containing nine patient samples and one pooled control sample. The pooled control sample consisted of a pool of peptides from all patient samples. The peptide samples derived from individual patients were then individually labelled using the TMT10plex mass tag kit (Thermo scientific, USA) according to manufacturer's instructions, with one isobaric tag being exclusively used to label the pooled control sample. The labelled peptides for each 10plex were subsequently combined to generate 10 individual pools. The labelled peptide pools were desalted using P10 C18 pipette ZipTips (Millipore, USA) according to the manufacturer's instructions. Eluted peptides were dried in a Speedvac concentrator (Thermo Scientific, USA). Peptides (8 μl) were loaded using a Dionex Ultimate 3000 nanoflow ultra-high-pressure liquid chromatography system (Thermo Scientific, USA) on to a 75µm x 2 cm C18 trap column (Thermo Scientific, USA) and separated on a 75µm x 50 cm C18 reverse-phase analytical column (Thermo Scientific) at heated at 40°C. For LFQ protein quantification; elution was carried out with mobile phase B (80% acetonitrile with 0.1% formic acid) gradient (4 to 30%) over 310 min at a flow rate of 0.25 μl/min.