Synapt g1 mass spectrometer

The microfluidic chip was made on a poly (methyl methacrylate) (PMMA) substrate with dimensions of 5.1 cm × 2.0 cm × 1.2 cm and the proteolytic chamber was etched onto the chip using a VersaLaser engraver (Universal Laser, Scottsdale, AZ) as previously described [36 (link)]. The time-resolved electrospray ionization (TRESI) mixer was made by inserting a glass capillary (152 μm outer diameter) into a metal capillary (inner diameter 178 μm) to create an intercapillary space of 26.8 μm. The end of the glass capillary was sealed using the VersaLaser and a notch was cut 2 mm from the sealed end. For reaction quenching, a T-mixer with a dead volume of 51 nL was used to mix glacial acetic acid with deuterated protein. Pepsin-agarose beads were crosslinked as previously described [37 (link)]. The proteolytic chamber was filled with pepsin-agarose beads, and a 33G metal capillary was used as an outlet to the electrospray ionization source. Hamilton syringes were used to deliver reagents through glass capillaries using Pump 11 Elite infusion pumps (Harvard Apparatus, Holliston, MA). The device was placed at the front end of a Synapt G1 mass spectrometer (Waters, Mississauga, ON) for HDX-MS experiments.

Two μl of the same sample used in the crystallization experiments was diluted in 50 μl of 50 mM ammonium bicarbonate pH 7.5. The sample was subsequently digested with trypsin (Porcine, sequencing grade, Promega, Madison, WI) O/N at 37 °C using an enzyme to protein ratio 1:20. Formic acid was added to a final v/v concentration of 0.1% before the liquid chromatography mass spectrometry analysis. The digest was separated using an ultra performance Nano Acquity system coupled to a Synapt G1 mass spectrometer (Waters, Milford, MA). The peptides were separated on an HSS T3 (75 μm × 250 μm, 1.8 μm particles) column (Waters) with a gradient of 3–40% of buffer A (0.1% formic acid) and buffer B (100% acetonitrile with 0.1% formic acid). The spectra were acquired in Liquid-chromatography mass spectrometry in elevated energy mode (LCMS^E) mode (alternating low and high collision energy) with mass range from 125 to 2,000 m/z using a collision energy ramp (10–40 V)61 (link). The data were analysed using the Proteinlynx Global Server V2.5 platform (Waters) against UniProt database using C-mannosylation as a variable modification.

Nuclear magnetic resonance (NMR) spectra were recorded in D₂O, CD₃OD, or CDCl₃ at 25°C using Bruker Avance 500 MHz, Varian Inova 500 MHz, and Agilent 600 MHz (3 mm cold probe) instruments, as indicated on the spectra in the Supplementary Figures S1–S4 and Supplementary Tables S1–S3. Spectra run in D₂O were not re-referenced. Spectra run in CD₃OD were referenced to the residual solvent peaks at 3.31 ppm and 49.0 ppm for ¹H and ¹³C NMR, respectively. Spectra run in CDCl₃ were referenced to residual solvent peaks at 7.25 and 77.0 ppm for for ¹H and ¹³C NMR, respectively. Accurate-mass MS data were recorded on a Waters Synapt G1 mass spectrometer after calibration with a sodium formate solution with a mass range from 100 to 2000 m/z. System suitability was obtained during each run, in which the difference between the calculated and observed m/z values were less than 5 ppm for each of four standard compounds, with m/z ranging from 152 to 609.

Soluble protein extracts were prepared as described previously5 (link)61 (link) in a buffer containing 30 mM Tris, 4 mM EDTA (called TA buffer), 1 mM NAD, 20% glycerol and 40 μg mL⁻¹ protease inhibitor (Sigma). The protein concentration was assayed using the Bio-Rad (Hercules, CA, USA) reagent using bovine serum albumin as a standard.
Extracts were incubated for 15 min at 80 °C with 10% sodium dodecyl sulfate (SDS), 10 mM DTT, 20% glycerol, 0.2 M Tris and 0.05% bromothymol blue. Protein migration was performed on 12% polyacrylamide gel Mini-PROTEAN^® Tetra Cell (Biorad, Hercules, USA). Gels were either stained with Coomassie blue to determine any differential pattern at low and high CO₂ or the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, stained with red Ponceau, and used for N-terminal sequencing. LCIP63 was identified using a Synapt G1 mass spectrometer (Waters, Manchester, UK) coupled to a nano flow UPLC nanoAcquity (Waters). Spectra and protein search were processed by the PLGS 3.0.1 software (Waters) with the same search parameters as described below. N-terminal sequence determination was performed by Edman degradation using an automatic sequencer (Procise 494, Applied Biosystems). Mature sequence of LCIP63 was analysed using Hectar (http://webtools.sb-roscoff.fr/) to identify the location of the protein.

Crystals of Cry11Aa were centrifuged for 5 min at 5000 × g during the buffer wash and washed twice with ammonium acetate buffer (pH adjusted to 6.4 with acetic acid). Pelleted crystals were then dissolved in ammonium acetate buffer (pH adjusted to 11.5 using ammonium hydroxide). Gold-coated capillary emitters were prepared as previously described and used to load the protein sample^{105 (link)}. The sample was analyzed on a Synapt G1 mass spectrometer (Waters Corporation). The instrument was tuned to preserve non-covalent interactions. Briefly, the capillary voltage was set to 1.60 kV, the sampling cone voltage was 20 V, the extraction cone voltage was 5 V, the source temperature was 80 °C, the trap transfer collision energy was 10 V, and the trap collision energy (CE) was set at 30 V. For MS/MS characterization, a particular charge state was isolated in the quadrupole and the complex was dissociated by application of 200 V of CE. The data collected were deconvoluted and analyzed using UniDec^{106 (link)}.

UV spectra were recorded on a UV-1700 PharmaSpec Shimadzu spectrophotometer (Shimadzu Corp., Kyoto, Japan) in MeOH. IR spectra were measured with a PerkinElmer Spectrum 2000 FT-IR spectrometer (PerkinElmer Corp., Waltham, MA, USA). NMR experiments were conducted on a Varian Mercury FT-NMR 400 MHz spectrometer (Agilent Corp., Santa Clara, CA, USA) using tetramethylsilane (TMS) as an internal standard. High Resolution Electrospray Ionization Mass Spectra (HRESIMS) and Electrospray Ionization Mass Spectra (ESIMS) were determined on Waters SYNAPT G1 mass spectrometer (Waters Corp., Milford, MA, USA).

UPLC-ESI MS analysis was performed on a UPLC-Acquity chromatograph (Waters) coupled to an SYNAPT G1 mass spectrometer (Waters). Aliquots of 10 μL of each sample were analyzed on an ACQUITY UPLC CSH C18 column (1.7 μm; 2.1 × 50 mm; Waters). The mobile phase consisted of a mixture of solvent A (water with 0.1% v/v formic acid) and solvent B (acetonitrile with 0.1% v/v formic acid). The column temperature was 30 °C. The flow was adjusted continuously at 3 µL/min.
The acquisition of ESI MS was performed in positive mode, and the conditions were as follows: capillary voltage, 2.2 kV; cone voltage, 30 V, and exhaust voltage, 4 V.