Jupiter c18

The syntheses and characterization of SBK2-Tris-(Gd-DOTA)₃ and scrambled-Tris-(Gd-DOTA)₃ agents have been described previously [8 (link)]. Briefly, the peptides were synthesized using conventional solid-phase synthetic methods and FMOC-protected amino acids. Peptide purity was assessed using LC-MS/MS on a Thermo Finnigan LTQ Linear ion trap mass spectrometer with a Phenomenex Jupiter C18 reversed-phase capillary chromatography column. Peptides were conjugated to maleimido-tris-propargyl by reacting the N-terminal cysteine with the maleimide group. A copper-catalyzed azide-alkyne cycloaddition reaction was then used to couple azido-(Gd-DOTA) to the free alkyne groups of the tris-propargyl peptides. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF; Autoflex Speed, Bruker Corp., Billerica, MA, USA) was used to monitor the conjugation reaction of azido-(Gd-DOTA) to the peptide-tris-propargyl moieties. Gadolinium (Gd) content was measured using inductively coupled plasma optical emission spectroscopy (ICP-OES) (Agilent 730 Axial ICP-OES; Agilent Technologies, Wilmington, DE, USA). T₁ relaxation constants for the agents were measured on the Bruker Biospec 9.4T MRI scanner (Bruker Corp., Billerica, MA, USA) at 37 °C.

Peptides were reconstituted in water containing 0.2% formic acid and analysed by nanoflow-LC-MS/MS using an Orbitrap Fusion Mass spectrometer (Thermo Scientific) coupled to a Proxeon Easy-nLC 1000. Samples were injected on a 300 μm ID × 5 mm trap and separated on a 150 μm × 20 cm nano LC column (Jupiter C18, 3 μm, 300 A, Phenomenex). The separation was performed on a linear gradient from 7 to 30% acetonitrile, 0.2% formic acid over 105 minutes at 600 nl per min. Full MS scans were acquired from m/z 350 to m/z 1,500 at resolution 120,000 at m/z 200, with a target AGC of 1E6 and a maximum injection time of 200 ms. MS/MS scans were acquired in HCD mode with a normalized collision energy of 25 and resolution 30,000 using a Top 3 s method, with a target AGC of 5E3 and a maximum injection time of 3,000 ms. The MS/MS triggering threshold was set at 1E5 and the dynamic exclusion of previously acquired precursor was enabled for 20 s within a mass range of ±0.8 Da.

One milligram of venom was subjected to reverse phase HPLC using a Waters system, Empower software, and a Phenomenex Jupiter C₁₈ (250 × 4.6 mm, 5 μm, 300 Å pore size) column as outlined in Smith and Mackessy [88 (link)]. Protein/peptide was detected at 280 nm and 220 nm with a Waters 2487 Dual λ Absorbance Detector. Fractions corresponding to each peak were then frozen at − 80 °C overnight, lyophilized and then analyzed along with 20 μg crude venom via SDS-PAGE as described [88 (link)] in order to determine peak complexity, mass and toxin families [43 (link)]. Percent peak area at 280 nm was recorded as a proxy for relative toxin abundance.

Bronchoalveolar lavage fluid (BAL) was collected from male and female C57BL/6 mice (4–5 months old) according to the procedure described in [31 (link),32 (link)]. Mice were euthanized by means of CO₂ narcosis; a small incision at the level of the neck was made and a blunt needle connected to a syringe was inserted into the trachea. Then, lungs were washed with 1 mL of sterile PBS. BAL samples from each mouse were pooled, centrifuged at 1000 rpm (corresponding to 0.1× g) for 5 min, and stored at −80 °C. The supernatant was used for stability tests.
Peptides were dissolved in 400 µL of PBS at a concentration of 300 µM, and added to an equal volume of BAL at a final concentration of 150 µM. At different time points, 150 μL aliquots were withdrawn and added to 500 µL of acetonitrile, and then centrifuged. The supernatants were diluted with 160 μL of 0.1% trifluoroacetic acid (TFA)—water and analyzed by RP-HPLC and mass spectrometry. Liquid chromatography was performed on a Phenomenex Jupiter C₁₈ analytical column (300 Å, 5 μm, 250 × 4.6 mm) in a 30 min gradient, using 0.1% TFA in water as solvent A and acetonitrile as solvent B. Mass spectrometry analysis of diluted samples was performed with a Bruker Daltonic-ultraflex-matrix-assisted laser desorption ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometer.

The lyophilized crude N. atra venom was dissolved in water and centrifuged at 10,000× g for 10 min. The amount of protein in the venom was determined via a BCA Protein Assay kit (Pierce^TM, Thermo Scientific). The supernatants were diluted and further purified by size exclusion chromatography. The purified venom proteins, NTXs, were isolated from the crude venom following the procedure described by Huang et al. [18 (link)]. All the venom protein components other than the NTXs were combined and dissolved in PBS, creating the deNTXs. The crude venom and deNTXs were loaded onto a Phenomenex Jupiter^® C18 (250 × 4.6 mm, 5 µm particle size, 300 Å pore size) column with an ultraperformance liquid chromatography (UPLC) system (LC-20ADXR, Shimadzu, Kyoto, Japan) equipped with a DAD detector (SPD-M20A, Shimadzu, Kyoto, Japan) and autosampler (SIL-20ACXR, Shimadzu, Kyoto, Japan). The venom components were eluted at 1 mL/min with a linear gradient of 0.1% TFA in water (Solvent A) and 0.1% TFA in 100% ACN (Solvent B) (2% B for 5 min, followed by 2–10% B for 2 min, 10–16% B for 6 min, 16–28% B for 2 min and 28–65% B for 37 min) [18 (link)]. Protein elution was monitored at 215 nm (absorption wavelength for peptide bonds). The relative abundance (expressed as the percentage of the total venom protein) of each protein family was estimated as described by Huang et al. [18 (link)].