The venom apparatus of one “nonmilked” M. gulosa specimen was divided into four gland sections, the venom reservoir, and the venom duct. Each sample was mixed and then centrifuged to pellet tissue. The venom gland samples were concentrated using a C18 ZipTip (Thermo Fisher Scientific): 20 μl was loaded onto the ZipTip, washed with 10 μl of 0.1% trifluoroacetic acid (TFA), and eluted in approximately 2 μl of ACN. Venom reservoir, venom duct, and Dufour’s gland samples and an aliquot of injected venom were diluted 1:10. One microliter of each sample was mixed with 2 μl of diluted α-cyano-4-hydroxycinnamic acid solution [stored as an acetone-saturated solution and diluted 1 in 10 with a solution of ethanol/acetone/0.1% TFA (6:3:1) as a working solution] and spotted onto a polished steel target. MALDI spectra were acquired using a Bruker Autoflex Speed MALDI-TOF/TOF system (Bruker Daltonics Inc.) in linear positive mode at 2000 Hz, with an m/z range from 1000 to 15,000. Ten spectra of 500 shots each were saved. The group of 10 spectra were loaded into ClinProt Tools version 3.0 (Bruker Daltonics Inc.) as a separate group and observed as a gel view, with the averaged spectrum for the entire data set produced after recalibration of the entire loaded sample cohort.

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