Agilent 3100 offgel fractionator system

For proteomic analysis, a total of 18 adult female B. malayi were collected by washing out the peritoneal cavity of Mongolian jirds (Meriones unguiculatus) with warm RPMI 1640 media not containing FBS. Worms were processed by sonication (in 25 mM ammonium bicarbonate) at 70% amplitude for 30 s with 30 s rest on ice (a total of three cycles). Proteins were treated with the surfactant 0.1% (v/v) RapiGestTM (Waters) at 80°C for 10 min followed by reduction with diothiothreitol (DTT) at a final concentration of 3 mM (60°C for 10 min) and alkylation with iodoacetamide (IAA) at a final concentration of 50 mM (room temp, in the dark, 1 h). The enzyme trypsin (sequencing grade, Promega) was added at an enzyme: substrate ratio of 1:50 and incubated overnight at 37°C. The surfactant was inactivated the following day by treatment with 0.1% trifluoroacetic acid (TFA) (37°C for 1 h), peptides were recovered following centrifugation at 13,0000 g. A proportion of unfractionated peptide sample was retained for mass spectrometric analysis. A total of 150 μg of digested protein material was subjected to extensive fractionation over the pH range 3-10 using the Agilent 3100 OFFGEL fractionator system as per manufacturer’s instructions.

For TMT labeling, digested peptides were resuspended in 200 mM triethyl ammonium bicarbonate (TEAB, Sigma-Aldrich). TMT™ Isobaric Mass Tagging reagent (Thermo Scientific) were dissolved in anhydrous acetonitrile and added to each sample. After incubation for 1 h at room temperature, the reaction was quenched with 5% hydroxylamine. The labeled samples were pooled into one tubes and separated with an Agilent 3100 OFF-GEL fractionator system (Agilent Technologies, Santa Clara, CA, USA) according to manufacturer's instructions. Finally, resulting samples were desalted using C18 Spin-Column (Thermo Scietific).