Impact 2

The untargeted analysis was performed using a Bruker Elute UHPLC system with Hystar 3.3 software and an ultra-high-resolution mass spectrometer Bruker Impact II (60,000 + resolution version; Bruker Daltonik GmbH) ESI QTOF-MS with Data Analysis 4.2 (Bruker Daltonik GmbH) and Metaboscape (ver. 2022b). Metabolite separation was achieved with a gradient of mobile phases using a Waters UPLC column ACQUITY BEH (C18 silica, 1.7 μm particles, 50 × 2.1 mm) with a compatible column guard was used for all analyses. Further details are described in our previous publication^{35 (link)} and supplementary information 1 (section S1).

Each sample was analyzed on a Bruker Impact II (Bruker Daltonics, Germany) connected to a Dionex UHPLC system (Thermo Fisher Scientific). The UHPLC was equipped with a trap column (Acclaim PepMap 100, 75 μm × 2 cm, nanoviper, C₁₈, 3 μm, 100 Å; Thermo Fisher Scientific) and an analytical column (PepMap RSLC C₁₈, 2 μm, 100 Å, 75 μm × 50 cm; Thermo Fisher Scientific). Mobile‐phase buffers for nLC separation consisted of 5% ACN/0.1% FA in water (solvent A) and 95% ACN/0.1% FA (solvent B). The peptides were eluted during a 2 h gradient and directly sprayed into the mass spectrometer. The flow rate was set at 400 nl/min, and the LC gradient was as follows: 4% solvent B within 5 min, 4–32% solvent B within 90 min, 32–95% solvent B within 1 min, 100% B for 10 min and down to 2% solvent B within 1 min following equilibration at 2% solvent B for 13 min. Nano spray was achieved with an applied voltage of 1.6 kV using a Captive Spray source (Bruker Daltonics, Germany) connected to a NanoBooster filled with 100% ACN. The mass spectrometer was programmed in a data‐dependent acquisition mode (cycle time set to 3 s) and was configured to perform a survey scan from 150 to 2200 m/z (1 Hz). MS2 scans were acquired in dynamic mode (2500 cts = 8 Hz to 25,000 cts = 32 Hz) of ions with charge state between 2 and 5 with a smart exclusion (5×) set to 30 s.

A. zaruma venom extract was analyzed by a bottom-up proteomic approach using positive electrospray ionization (ESI) performed on quadrupole time-of-flight (Q-TOF) mass spectrometer (Bruker Impact II, Bruker Daltonics, Germany). Samples were prepared as follows: lyophilized crude venom extract of A. zaruma, was resuspended in water for a total stock concentration of 10 µg/µL. 100 µL of the stock solution was dissolved in 200 µL of 100% acetonitrile and cleared by centrifugation at 10 rpm for 5 min. 300 µL of supernatant was dissolved in 700 µL ddH₂0 to get a final concentration of 1 mg/mL A. zaruma venom extract (1/10 dilution). Different concentrations for histamine samples were prepared according to the same protocol: 0.1 µM, 1 µM, 10 µM and 100 µM. The Q-TOF system was operated in positive mode using Bruker TargetScreener HR 4.0. This program includes hardware, column and methods with a total analysis time including chromatographic separation and accurate mass detection of 20 min (12 ). During data acquisition, both MS and MS/MS full scan mode datasets were obtained. The MS raw data were obtained in format from Bruker Compass Data Analysis Viewer version 5.2 (Bruker Daltonics, Germany).

The peptides Drosocin (Anaspec, United States), H2686, H8390, Amyloid β (10–20), Neuromedin U-25, MOG (Bachem, Switzerland), and Insulin Chain B Oxidized (IOB; Sigma, United States) were incubated with MdpL (18 h, 37°C) in 0.1 M sodium phosphate, pH 6.5, with 20 mM L-cys to detect any preferential sites permitting MdpL hydrolysis. Fragments were separated using LC (Agilent Technologies 1,290 Infinity, C18) and analyzed by MS/MS (Bruker Impact II). MdpL hydrolysis sites were mapped using the Bruker Compass Data Analysis 5.2 and BioPharma Compass 4.0 software. Since the generated peptide ions have different flight characteristics during an ESI-TOF-MS run, the prevalence of amino acids was not based on area under the curve calculations. A sequence logo (WebLogo 3, University of Berkeley) illustrating the most occurring amino acid +/− three positions from the MdpL hydrolytic site was created by entering the 89 detected high score (>30) peptides generated from the hydrolysis of Neuromedin U-25, MOG, and Insulin Chain B Oxidized. Drosocin, H2686, H8390, and Amyloid β were not hydrolyzed by MdpL and thus were not included in the data acquisition. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2021 (link)) partner repository with the dataset identifier PXD039681.