Xcalibur 2.2 software suite

Native MS [44 (link)] was used to investigate complexes formed between Nt17 peptide and various lipid vesicles [45 (link)]. Lipid vesicles were formed as previously described, but films were reconstituted in HPLC-grade water instead of tris buffer. Nt17 peptide (20 μM) was incubated with lipid vesicles (20:1 lipid:peptide) for 5 h at 37 °C. Samples were analyzed using a Q Exactive Hybrid Quadrupole mass spectrometer (Thermo Fischer, San Jose, CA) equipped with a commercial HESI source. Spectra were recorded in positive-ion mode over a mass-to-charge (m/z) ratio range of 400 to 4,000, and samples that included anionic lipids were also recorded in negative-ion mode. Samples were infused at a rate of 10 uL/min and the needle was biased at 3,500 V relative to the mass spectrometer inlet. The parameters for the MS instrument were: 400 °C for the capillary inlet temperature, 30 °C for the analyzer temperature, 80 V for the S-lens assembly, 400 ms for the maximum injection time, 1 × 10⁶ for the AGC, and 70,000 for the MS resolution. Each spectrum was recorded in triplicate for 90 s each, and the data was analyzed using the Xcalibur 2.2 software suite (Thermo Scientific).

Samples were analyzed using cVSSI devices and an LTQ-XL mass spectrometer. Spectra were collected for at least 30 sec each in positive ion mode over a mass-to-charge (m/z) range of 275 to 1,000. Samples were infused at a flow rate of 10 μL/min with 1.8 kV applied to the platinum wire. After a precursor ion spectrum for each sample was collected, collision-induced dissociation (CID) using a normalized collision energy of 30 was performed on the peak(s) corresponding to the +2 charge state of Nt17. Data were analyzed using the Xcalibur 2.2 software suite (Thermo Scientific).

Synthetic htt-exon1 mimic peptide (Nt17-Q₃₅-P₁₀) was prepared as described previously. Dried peptide films were each solubilized in TFA water (pH 3). Pepsin (porcine) was then added to the synthetic peptide (20:1 peptide to enzyme ratio) and immediately adjusted to a pH of 2 using formic acid. Peptides were incubated with pepsin at 37 °C for approximately 36 h prior to LC-MS analysis. Samples of undigested peptide were also prepared by removing some of the solubilized synthetic peptide prior to addition of pepsin. Undigested peptide was diluted to appropriate stock concentration using water and acetonitrile, then adjusted to a pH of 2 using formic acid to prevent aggregation.
For peptide analysis, a Q-Exactive hybrid quadrupole Orbitrap (Thermo Fisher, San Jose, CA) mass spectrometer operated in positive ion mode was used over a mass-to-charge (m/z) range of 300 to 1,500. The resolving power was set to 70,000 and the AGC target was set at 1×10⁶. The inlet capillary temperature was maintained at 250 °C. The sheath gas flow rate was 48, the auxiliary gas flow rate was 11, and the sweep gas flow rate was 2.5. The S-lens RF level was set at 50 V, and the voltage applied to the HESI source was +3.5 kV. Samples were injected at a volume of 20 μL and a flow rate of 30 μL/min. Data were analyzed using the Xcalibur 2.2 software suite (Thermo Scientific).