Development of Onchocerca volvulus in humanized NSG mice and detection of parasite biomarkers in urine and serum

Patton, John B.; Bennuru, Sasisekhar; Eberhard, Mark L.; Hess, Jessica A.; Torigian, April; Lustigman, Sara; Nutman, Thomas B.; Abraham, David

Serum and urine were collected and frozen as terminal procedures during necropsy. Serum was thawed on ice and 25 μL removed for processing from each mouse. Sera from 4 mice from each group/strain were pooled for maximizing the protein identifications. Abundant proteins were depleted using an affinity chromatography (MARS-Ms-3, Agilent) according to the manufacturer's directions. Urine was thawed on ice, centrifuged and then filtered through a 0.22 μM filter (Corning). Serum and urine samples were prepared for mass spectrometry by digestion using the filter-assisted sample preparation (FASP) method [49] . Briefly, the samples brought to 1% sodium deoxycholate (SDC), 50 mM Tris-HCl, pH 7.6, 3 mM dithiothreitol, sonicated briefly, and incubated in a Thermo-Mixer at 90 o C, 1,000 RPM for 20 min. Samples were centrifuged to clarify and the supernatant was transferred to a passivated 30 kD MWCO device (Millipore, Merck KGaA, Darmstadt, Germany) and centrifuged at 13,000g for 30 min. The remaining sample was buffer exchanged with 1% SDC, 100 mM Tris-HCl, pH 7.6, then alkylated with 15 mM iodoacetamide. The SDC concentration was reduced to 0.1%. Samples were digested using trypsin at an enzyme to substrate ratio of 1:100, overnight, at 37 o C in a thermo-mixer at 1,000 RPM. Digested peptides were collected by centrifugation and the filter washed with 0.5 NaCl to elute electrostatically bound peptides. Digested peptides were desalted using reversed phase stop-and-go extraction tips [50] . Peptides were eluted with 80% acetonitrile, 0.5% formic acid and lyophilized in a SpeedVac (Thermo Savant, Holbrook, NY) to near dryness, approximately 1 h.