Deep Proteoform Sequencing with Top-Down Direct Mass Technology

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Conventional top-down mass spectrometry faces limitations in resolving overlapping isotopic distributions, hindering deep proteoform sequencing.
• Orbitrap mass spectrometers offer advanced capabilities for protein analysis, but further improvements in sensitivity, resolution, and mass range are desirable.

Purpose of the Study:

• To present an end-to-end workflow for deep proteoform sequencing using top-down mass spectrometry with Individual Ion Mass Spectrometry (I²MS) and Direct Mass Technology mode (DMTm).
• To demonstrate the enhanced performance of DMTm compared to conventional top-down mass spectrometry methods across various fragmentation modes.
• To introduce Proteoform Studio, a software platform for optimized ion processing and data integration to achieve comprehensive sequence coverage.

Main Methods:

• Utilized Individual Ion Mass Spectrometry (I²MS) with Direct Mass Technology mode (DMTm) on an Orbitrap mass spectrometer.
• Assigned charge of individual fragment ions and converted spectra from m/z to the mass domain to resolve isotopic distributions.
• Employed multiple fragmentation modes and integrated data from DMTm and conventional top-down methods using Proteoform Studio.

Main Results:

• Top-down DMTm significantly outperformed conventional methods, increasing sequence coverage for a glycosylated antibody heavy chain from 27.5% to 83.3% in 10 minutes.
• Achieved near-complete coverage (>95%) of the internal region of a large protein, a region previously difficult to characterize.
• Combined DMTm and conventional top-down data yielded 90.2% heavy chain sequence coverage, demonstrating complementary fragmentation patterns.

Conclusions:

• The developed top-down DMTm workflow enables substantially deeper proteoform sequencing with improved sensitivity, resolution, and mass range.
• Proteoform Studio facilitates comprehensive sequence coverage by optimizing ion processing and integrating data from complementary methods.
• This straightforward and complete workflow confidently defines proteoforms in biological systems and aids biotherapeutic development.