Optimized Low-Field Differential Ion Mobility Separations with High-Resolution Mass Spectrometry for Top-Down...

Area of Science:

• Analytical Chemistry
• Biophysical Chemistry
• Proteomics

Background:

• Ion mobility spectrometry coupled with mass spectrometry (IMS/MS) is crucial for analyzing complex biological mixtures, distinguishing isomers, and determining molecular geometries.
• Traditional IMS methods (linear and FAIMS) have limitations in analyzing larger or more complex molecules, necessitating advancements for high-resolution applications in proteomics and structural biology.
• Fourier-Transform (FT) MS coupled with IMS is essential for handling increasingly heavy macromolecules and complex samples requiring high resolution and accuracy.

Purpose of the Study:

• To integrate a novel nonlinear low-field differential (LOD) IMS technique with Orbitrap MS/MS for enhanced analysis of smaller proteins.
• To explore the capabilities of LODIMS in separating and characterizing noncovalent forms of Concanavalin A (ConA).
• To identify novel proteoforms and assess molecular properties like directional cross sections and dipole moments of ConA.

Main Methods:

• Integration of LODIMS using bisinusoidal or augmented flexible rectangular waveforms with Orbitrap MS and MS/MS.
• Analysis of Concanavalin A (ConA, 25.6 kDa) to assess separation of intact and fragmented forms.
• Characterization of molecular properties including directional cross sections and dipole moments using LODIMS waveforms.

Main Results:

• The LODIMS/MS platform successfully disentangled isotopic envelopes and separated intact ConA from its noncovalent fragments across various charge states.
• Consistent directional cross sections and dipole moments were obtained using rectangular and bis waveforms, indicating effective alignment of smaller proteins.
• Discovery of two novel ConA forms resulting from endogenous cleavage at N¹⁶² (in addition to the known N¹¹⁸ cleavage site).

Conclusions:

• LODIMS is capable of aligning smaller proteins, enabling reliable assessment of their directional cross sections and dipole moments.
• The developed LODIMS/Orbitrap MS platform facilitates top-down proteomics by improving the detection and characterization of intact proteins and their isoforms.
• This study demonstrates the potential of LODIMS for discovering novel proteoforms and advancing structural biology and proteomics research.