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

Resolution, 17000 (35000 for labelled peptides); AGC target, 5e4; maximum IT, 120 ms ; isolation window, 1.6 m/z. MS data were acquired by data dependent acquisition where the top 12 (15 for labelled peptides) most intense precursor ions in positive mode were selected for ms^2 Higher-energy C-trap dissociation fragmentation which were subsequently excluded for the next 45 s following fragmentation event. Charge exclusion was set to ignore peptide spectrum matches that were unassigned, singly charged, and those with ≥+8 charges. Raw mass spectrometer files were analysed by MaxQuant software version 1.6.0.1. by searching against the human Uniprot FASTA database Eluted peptides were dried in a Speedvac concentrator (Thermo Scientific, USA). Peptides (8 μl) were loaded using a Dionex Ultimate 3000 nano-flow 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. Each LC run was finished by washout with 98% B for 10 min and re-equilibration in 2% B for 30 min. Five blanks of 40 min each were run on the column between each injection comprising of two wash cycles with 90% B and an equilibration phase of 15 min to avoid sample carryover. Peptides were measured using a Q Exactive Orbitrap mass spectrometer (Thermo Scientific, USA) coupled to the chromatography system via a nano-electrospray ion source (Thermo Scientific). On the Q Exactive , the ms^1 settings for peptides were: Resolution, 70000; AGC target, 3e6; maximum IT, 120 ms; scan range, 400-1800 m/z; while the ms^2 settings for fragmentation spectra of peptides were: Resolution, 17000 (35000 for labelled peptides); AGC target, 5e4; maximum IT, 120 ms ; isolation window, 1.6 m/z. MS data were acquired by data dependent acquisition where the top 12 (15 for labelled peptides) most intense precursor ions in positive mode were selected for ms^2 Higher-energy C-trap dissociation fragmentation which were subsequently excluded for the next 45 s following fragmentation event. Charge exclusion was set to ignore peptide spectrum matches that were unassigned, singly charged, and those with ≥+8 charges. Raw mass spectrometer files were analysed by MaxQuant software version 1.6.0.1. by searching against the human Uniprot FASTA database (downloaded February 2014) using the Andromeda search engine.