Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers

RATIONALE: The collision cross-section (O) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known O. The T-wave O-values depend strongly on instrument parameters and calibrant selection. Optimization of instrument parameters and calibration standards are crucial for obtaining accurate T-wave O-values. METHODS: Human insulin and the fast-acting insulin aspart under native-like conditions (ammonium acetate, physiological pH) were analyzed on Waters SYNAPT G1 and G2 HDMS instruments. The calibrated T-wave O-values of insulin monomer, dimer, and hexamer ions were measured using many different combinations of denatured and native-like calibrants (masses between 2.85 and 256 kDa) and T-wave conditions. Drift-tube O-values were obtained on a modified SYNAPT G1. RESULTS: Insulin T-wave O-values were measured at 26 combinations of T-wave velocity and amplitude. Optimal sets of calibrants were identified that yield O-values with minimal dependence on T-wave conditions and calibration plots with high R2-values. The T-wave O-values determined under conditions satisfying these criteria had absolute errors < 2%. Structural differences between human insulin and fast-acting insulin aspart were probed with IM-MS. Insulin aspart monomers have increased flexibility, while hexamers are more compact than human insulin. CONCLUSIONS: Accurate T-wave O-values that are indistinguishable from drift-tube values are obtained when using (1) native-like calibrants with masses that closely bracket that of the analyte, (2) T-wave velocities that maximize the R2 of the calibration plot for those calibrants, and (3) at least three replicates at T-wave velocities that yield calibration plots with high R2. Copyright (C) 2012 John Wiley & Sons, Ltd.