Synapt g2 hdms

All experiments were performed on a Waters Synapt G2 HDMS instrument equipped with a radio-frequency (rf) confining ion mobility drift cell [37 (link)] and ion/ion reaction capabilities [38 ]. CAPTR was performed as described previously [33 (link)]. Briefly, for 0.1 s the [M-F]⁻ fragments of perfluoro-1,3-dimethylcyclohexane (PDCH) were produced, quadrupole selected, and accumulated in the Trap Cell of the instrument. [PDCH−F]⁻ reacts exclusively through proton transfer (Reaction 2) [29 (link), 39 , 40 (link)]. Following anion accumulation, the instrument was switched into positive mode for 5 to 10 s, during which time a single charge state of cytochrome c was quadrupole selected and transferred into the Trap Cell for CAPTR. Every 22 ms, CAPTR products and unreacted precursor ions were injected into the rf-confining drift cell [37 (link)] with a 212 V drift voltage and filled with 2.0 mbar helium. Based on the measured arrival-time distributions, the apparent Ω distributions were calculated using a method described in the Electronic Supplementary Material that is identical to that used previously for the CAPTR products of denatured ubiquitin [33 (link)].

A quadrupole ion mobility-time of flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA) was used for all ion mobility experiments. 5uL of buffer-exchanged protein solution (20uM) was transferred to a gold-coated borosilicate capillary (0.78mm i.d., Harvard Apparatus, Holliston, MA) for direct infusion. Instrumental settings were optimized to preserve intact protein complexes prior to activation: capillary voltage 1.5 kV, sample cone 40 V, extraction cone 0 V. Gas flows (mL/min): source: 50, trap: 6 (Avidin) or 8 (ADH, Ovalbumin), helium cell: 200, IM separation: 90. IMS traveling wave settings were the same for all proteins: wave velocity: 150 m/s, wave height: 20 V, IMS bias: 5 V. Backing pressure was set to 5.5 mbar (Avidin), or 8.0 mbar (ADH, Ovalbumin). A single charge state of each protein complex was selected and collisionally activated in the trap cell (trap collision voltage: Avidin, 140 V; Ovalbumin, 160 V; ADH, 200 V) prior to ion mobility separation. Trap (collision cell) pressures were 3.8 E-2 mbar (Avidin, Ovalbumin) or 4.4e-2 mbar (ADH). Time of flight pressure was 1.8 E-6 mbar for all analyses. Scans were combined for 30 seconds (Avidin) or 10 minutes (Avidin, ADH, Ovalbumin) to obtain sufficient signal to noise ratios.

PvDBP Region II and monoclonal antibodies were purified as described [29 (link)] and buffer exchanged into PBS. Stock antigen and antibodies solutions in PBS buffer were exchanged into 200 mM ammonium acetate solution (pH = 6.5–7). Before each experiment, the antigen and antibody were mixed and diluted to 5 μM and 6 μM, respectively. The sample solution was incubated at 25 ºC for 30 min before MS analysis.
Samples were analyzed using a quadrupole ion-mobility time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Protein and complex ions were generated in a nESI source in the positive-ion mode. The source parameters were optimized to provide gentle ESI conditions. The traveling-wave IM separator was operated at a N₂ pressure of ~1.5 mbar; the IMS wave velocity was 650 m/s; and the IMS wave height 40 V. The ToF-MS was operated over a m/z range of 100–15,000. For a CIU experiment, the trap-collision voltage applied to the ions in the traveling-wave-based ion trap situated prior to the IM separator was increased from 10 to 200 V in 10 V increments, and ion-mobility mass spectra were acquired at each increment.
Mass spectra were processed with Masslynx V4.1 software (Waters). Arrival time distributions at each collision voltage were extracted in the Drift Scope (Waters) and plotted as a 2D contour plot using Origin 2015 (OriginLab, Northampton, MA).

All data were acquired on a MALDI-IM-MS (Synapt G2 HDMS; Waters Corp., Milford, MA, USA) using a frequency-tripled Nd:YAG laser (wavelength=355 nm, pulse repetition frequency=1000.0 Hz). Data were acquired in Resolution mode and, unless otherwise indicated in selected measurements, over a mass range of 400 to 600m/z for sterol standards in characterization and development of the technique and a mass range of 480 to 510m/z for imaging-mode acquisition. Laser energy attenuation was 300 (arbitrary units) for cholesterol and 7-DHC standards and 250 for ionization directly from cells. Ion mobility separation was performed at a drift gas pressure of 2.30 Torr using nitrogen gas, a wave velocity of 650 m/s, and a wave height of 40.0 V.