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Probing Native Protein Conformations with IMgenius™

· Ion Mobility,Electrospray,Native Structure

Introduction:

Based on the current industry drive for accelerated drug development, there is a heightened need for screening native protein structures. Atmospheric pressure Ion Mobility Spectrometry (IMS) is an emerging analytical technology that addresses this challenge. IonDX Inc. has developed a bench-top analytical system, IMgenius™, based on this technology. Our patented spectrometer simultaneously measures five important attributes of proteinsand biotherapeutics: 

• Size distribution

• Structural conformation

• Stability, including aggregation

• Comparability 

• Purity 

IMgenius SystemTM and Methodology:

Charge-reduced electrospray: Desalted samples are electrosprayed at 0.3 uL/min followed by passing the electrospray droplets through a charge-reducing chamber where droplet charge is reduced to a single charge. Charge reduction is accomplished by exposing the droplets to bipolar air ions produced when alpha particles are emitted by a safely disposable sealed Polonium source. In this way protein ions never pass through high charge states and remain in their native conformation, allowing native conformations to be studied.

Atmospheric pressure ion mobility spectrometry: IMgenius determines the mobility (K) of a population of molecular ions as they travel through atmospheric pressure air and are detected as an ion current after they are deflected onto a pick-up electrode by voltage applied to a center rod. Small ions have higher mobility than larger ions because they experience less drag as they travel through a gas and therefore are deflected more easily.Inverse mobility (1/K) plots are easily interpreted as 1/K scales with molecular size. The width of a peak in an ion mobility spectrum correlates with molecular heterogeneity. Wider peaks indicate greater heterogenity. Peak position (modal 1/K value) indicates molecular size. 1/K values are directly convertible to collision cross section (CCS).

Native Conformation of Myoglobin:

Ko values for myoglobulin (blue) ref obtained with IM-MS show variation across ion charge states, even for an individual charge state, which makes it difficult to identify the native conformation. Our ion source produces lowly�charged ions (red) and when extrapolated towards z = 0, actually passes through 0. The zero intercept confirms the preservation of native conformations. See below for discussion of the Mason-Schamp equation. 

Native Confirmation of NISTmAb:

Ko values for NISTmAb (blue) ref obtained with IM-MS lead to a prediction of CCS = 68 nm2. This value is significantly less than 100 nm2 predicted from X-ray crystallography and attributable gas-phase collapse. conditions in our ion source and IMgenius minimize gas-phase collapse.

The Mason Schamp Equation provides an expression for determining CCS (sigma) from Ko. Plotting Ko vs z should be a straight line with a zero intercept. Plots with non-zero intercepts, as shown above, indicate the ions are stretched out and therefore not in their native conformation. Our data has a zero intercept and confirms our production of native conformations.

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