Agilent 6560

K-RAS and its mutants were fully loaded with GTP and analyzed using an Agilent 6560 ion mobility Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA).^{51 (link)} For native ion mobility mass spectrometry (IM-MS) measurements, ions were generated from a nanoelectrospray ionization source. Ions were then pulsed into the drift tube filled with ~3.95 Torr of nitrogen gas, where they traveled under the influence of a weak electric field (5 V cm⁻¹ Torr⁻¹). Ions exiting the drift tube were refocused by a rear ion funnel prior to QTOF MS detection, and their arrival times (t_A) recorded. CCS values were determined from first principles with data acquired at different drift voltages.^{52 (link)} Instrumental settings are listed in Tables S2 and S3. The Agilent IM-MS Browser software was utilized to extract arrival time distributions (ATDs), IMS data processing, and all stepped field CCS calculations.

Metabolite CCS values were calculated from analyte drift time using the single field method described in previous works.^{27 (link),31 (link)–33 (link)} Briefly, a set of calibrant molecules with well-characterized CCS (Agilent Tune Mix) were analyzed using identical instrument conditions as metabolite experiments (gas pressure, voltage, etc.). Drift times for calibrant ions were extracted to establish a calibration curve correlating drift time to CCS. Measured drift times for metabolites were then obtained from sample injections to calculate CCS values from the calibration curve. CCS values collected for features were subsequently compared with standards measured in previous IMS studies, here, the CCS Compendium (McLean Laboratory, Vanderbilt University)^{32 (link)} and metabolite values therein collected from the work of Nichols et al. on the same DTIMS platform.^{34 (link)} CCS reproducibility between laboratories using the Agilent 6560 is typically 0.5% RSD, so for this work, an “acceptable” CCS match was defined as <1.0% difference to provide sufficient tolerance in variation for single experiments.

The IMS resolving power (R_p) of the Agilent 6560 operating either in the normal “single pulse” mode or multiplexed mode is ~60 R_p (CCS/ΔCCS) and has been rigorously characterized.^{22 (link),23 (link),32 (link)} Current efforts to improve IMS resolving power (equivalently, selectivity) of the current instrumentation are focused on postacquisition enhancement of raw data, a process termed “high-resolution demultiplexing” (HRdm), which has also been evaluated previously.^{29 (link)} The process utilizes a Hadamard transform to enhance the IMS spectra and is only available for use when data are collected in multiplexed mode.^{33 (link)} For this work we utilized a beta version of the HRdm software (v. 2.0_B45E) for enhancing IMS separation of isobars/isomers, and the detailed settings are provided in the Supporting Information (Figure S2). Our results indicate that the HRdm process improves the resolving power from ~60 to between 100 and 200, depending on the ion drift time and signal saturation.