title: Traveling-wave ion mobility mass spectrometry of protein complexes: accurate calibrated collision cross-sections of human insulin oligomers.
abstract: BACKGROUND
  The collision cross-section (Ω) 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 Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization of instrument parameters and calibration standards are crucial for obtaining accurate T-wave Ω-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 Ω-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 Ω-values were obtained on a modified SYNAPT G1.
  
  
  RESULTS
  Insulin T-wave Ω-values were measured at 26 combinations of T-wave velocity and amplitude. Optimal sets of calibrants were identified that yield Ω-values with minimal dependence on T-wave conditions and calibration plots with high R(2)-values. The T-wave Ω-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 Ω-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 R(2) of the calibration plot for those calibrants, and (3) at least three replicates at T-wave velocities that yield calibration plots with high R(2).
authors.affiliation: Diabetes Protein Engineering, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.
authors.forename: Rune
authors.initials: R
authors.lastname: Salbo
authors.forename: Matthew F
authors.initials: MF
authors.lastname: Bush
authors.forename: Helle
authors.initials: H
authors.lastname: Naver
authors.forename: Iain
authors.initials: I
authors.lastname: Campuzano
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Ingrid
authors.initials: I
authors.lastname: Pettersson
authors.forename: Thomas J D
authors.initials: TJ
authors.lastname: Jørgensen
authors.forename: Kim F
authors.initials: KF
authors.lastname: Haselmann
mesh_terms: D002138:Calibration; D006801:Humans; D061267:Insulin Aspart / Q000737:chemistry*; D061386:Insulin, Regular, Human / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods*; D021122:Protein Subunits / Q000737:chemistry
chemical_list: D061386:Insulin, Regular, Human; D007477:Ions; D021122:Protein Subunits; D061267:Insulin Aspart