Characterization of Gas-Phase Native(-like) Proteins Using Structures for Lossless Ion Manipulations

Area of Science:

• Analytical Chemistry
• Biophysical Chemistry
• Separation Science

Background:

• Structures for Lossless Ion Manipulations (SLIM) offer high-resolution ion separations in the gas phase.
• Previous SLIM applications have focused on various molecular classes, but protein analysis requires further development.

Purpose of the Study:

• To investigate multipass SLIM for separating gas-phase proteins in a near-native state.
• To analyze charge-state-dependent arrival time distributions and conformational changes in proteins.
• To validate SLIM-derived collision cross-section (CCS) values against drift tube ion mobility spectrometry (DTIMS).

Main Methods:

• Utilized multipass SLIM separations for carbonic anhydrase (CA), alcohol dehydrogenase (ADH), and apo-transferrin.
• Obtained experimental CCS values using calibration curves with Agilent Tune Mix ions.
• Deconvoluted multipass arrival time distributions (ATDs) into single-pass values for CCS determination.
• Compared SLIM-derived CCS values with DTIMS measurements.

Main Results:

• Baseline separation of CA charge states (z=9+ to 11+) achieved with 8-m single-pass SLIM.
• Improved mobility peak resolution for ADH (z=23+ to 25+) with extended path lengths up to 24-m.
• Observed transition from unimodal to multimodal CCS distributions for apo-transferrin (z=16+ to 18+).
• Demonstrated selective isolation of specific charge states (e.g., apo-transferrin z=17+) using 40-m multipass separations.
• SLIM separations showed minimal impact on protein structure and CCS values.

Conclusions:

• Multipass SLIM effectively separates gas-phase proteins in a near-native state, resolving charge-state-dependent structures.
• The technique provides accurate CCS values comparable to DTIMS.
• SLIM's capability for mobility-selective isolation opens avenues for native ion mobility applications and further SLIM-based manipulations.