Bradykinin

Highly deuterated peptides (Waters MassPREP Peptide Standard containing RASG-1, bradykinin, and angiotensin I and II) were prepared by dissolving the lyophilized peptides into D₂O that was adjusted to pD 2.5 with DCl. Peptides were allowed to deuterate at 20 °C for two hours before infusion directly into the instrument in 50:50 D₂O:acetonitrile using a syringe pump.
Labeled cytochrome c (462 µM stock solution in 20 mM Tris, 100 mM NaCl and 3 mM DTT) was diluted to usable concentrations of 64 and 12.8 µM for HPLC and UPLC, respectively. Deuterium exchange was initiated by adding a 15-fold excess of 99% deuterium oxide buffer (20 mM Tris, 100 mM NaCl and 3 mM DTT) at 21 °C. At each exchange-in time point an aliquot (100 picomoles for HPLC, 20 picomoles for UPLC) from the exchange reaction was transferred to a separate tube containing an equal volume of quench buffer (300 mM potassium phosphate, pH 2.6, H₂O). Quenched samples were immediately analyzed. Highly deuterated cytochrome c was prepared by diluting the stock solution 15-fold into D₂O pD 2.5, incubating at 37 °C for 6 hours and quenching as described above.

HIV consensus peptides were obtained from the NIH AIDS reagent program. 99.9% D₂O was obtained from Cambridge isotope labs (Tewksbury, MA, USA). Peptide P1 (HHHHHHIIKIIK-NH₂) was purchased from Anaspec (Fremont, CA, USA), and [Glu1]-fibrinopeptide B (GluFib), Angiotensin II, and bradykinin were purchased from Sigma Aldrich (St. Louis, MO, USA). Peptides were resuspended in Optima LC-MS water (Fisher Scientific, Pittsburgh, PA, USA)) for a stock concentration of 2 mg/mL. HIV Env gp140 SOSIP.664 expression, purification, and HDX-MS analysis were performed as described previously [21 (link)].
For LC-MS analyses, 20 pmol of denatured and maximally deuterated frozen aliquots of HIV Env gp140 SOSIP.664 were manually injected onto a Waters HDX manager coupled with a Synapt G2-Si. Samples were loaded onto custom prepared pepsin beads (POROS AL20; Life Technologies, Carlsbad, CA, USA) [26 ], at 150 µL/min and trapped onto a Vanguard 2.1 ×5 mm C18 BEH column for 5 min (Waters). Peptides were then separated with a 1 × 100 mm C18 BEH 1.7 µm column (Waters, Milford, MA, USA) using a linear gradient of 5 to 40% B (A: 2% ACN, 0.1% FA, 0.04% TFA; B: 100% ACN 0.1% FA) over 8 min at 40 µL/min. MS scans were collected every second using a source temperature of 80 °C and desolvation temperature of 150 °C. Other source parameters were left as default unless otherwise stated. A lock mass solution of leucine enkephalin was sampled every 90 s to maintain mass accuracy. A third switching valve was introduced to allow for stringent washing of the trap and pepsin columns between injections to minimize sample carryover (less than 2%) [15 (link), 16 (link)].
For direct infusions, Env peptides were diluted 50-fold into 95% D₂O containing 10 mM ammonium bicarbonate pH 8.0, and 0.02 mg/mL GluFib, Angiotensin II, or bradykinin. The solution was heated to 85 °C for 5 min for complete deuterium exchange and kept at 4 °C until MS analysis. Deuterium measurements were initiated by diluting 3 µLs of the deuterated peptide solution 100-fold into cold quench buffer (0.1% FA, 0.04% TFA, 100% H₂O, final pH 2.5). The solution was immediately loaded onto a 200 µL injection loop and pumped with the identical quench buffer isocratically at 40 µL/min to push the sample from the loop into the mass spectrometer. The injection loop and lines were kept under ice to minimize deuterium loss during the infusion. Source settings were adjusted to test effects on the deuterated spectra after the signal intensity stabilized. In-exchange measurements were performed identically except the stock peptide mixture was diluted 200-fold into ice-cold 98% D₂O, 0.1% FA, 0.04% TFA, and immediately injected onto the loop. Deuterium incorporation analysis and bimodal deconvolution were performed with HX-Express v2 [14 (link)].

Functional Ca²⁺ imaging on cultured mouse DRG neurons (2-3 DIV) was performed as described previously (Schnizler et al., 2008 (link)). Neurons on glass coverslips were incubated at room temperature (22°C) for 30 min with 2 μM of the AM form of the Ca²⁺-sensitive dye Fura-2 (Life Technologies). The coverslip was then placed in the recording chamber mounted on the stage of an inverted IX-71 microscope (Olympus Co., Tokyo, Japan) and washed for 10 min before the experiment began. Fluorescence was alternately excited at 340 nm and 380 nm (both 12 nm band pass) using the Polychrome IV monochromator (T.I.L.L. Photonics, Martinsried, Germany), via a 10× or 20× objective [numerical aperture (NA) 0.75; Olympus]. Emitted fluorescence was collected at 510 (80) nm using an IMAGO CCD camera (T.I.L.L. Photonics). Pairs of 340/380 nm images were sampled at 0.2 Hz. Bath application of capsaicin (15 sec) was performed twice with a 5 min interval, and NPs, prostaglandin E₂ (PGE₂), and bradykinin (BK) were applied for 5 min during this interval. The fluorescence ratio (R = F₃₄₀/F₃₈₀) was converted to [Ca²⁺]_i, as described earlier (Schnizler et al., 2008 (link)). Data were processed and analyzed using TILLvisION 4.0.1.2 (T.I.L.L. Photonics) and Origin 7.0 (Microcal, Northhampton, MA) software, and presented as mean ± SEM. All the Ca²⁺ imaging experiments with or without NP/PGE₂/BK treatments were performed in triplicate across three different batches of mouse DRG neuron cultures.