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

Naso-and oropharyngeal swab samples were centrifuged at 17,000xg for 10 mins at 4°C to obtain cell pellets which were washed once using PBS and lysed by bead-vortexing for 10 minutes in cell lysis buffer (RLT, Qiagen, Germany). Proteins (as well as DNA and RNA) were then extracted from the lysate using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Germany) following manufacturers instructions. The concentration of total protein obtained was determined using the Bradford assay (Bio-Rad, USA). Thirty micrograms (30µg) of total protein from each sample was then reduced with 10mM tris(2-carboxyethyl)phosphine (TCEP, Sigma-Aldrich, USA) at 55°C for 1h and subsequently alkylated with 18mM IAA (Sigma-Aldrich, USA) for 30 minutes at room temperature, while keeping the reaction protected from light. Proteins were precipitated overnight at -20°C with six volumes of pre-chilled (-20°C) acetone (Sigma-Aldrich, USA).