Expanding the mass range for UVPD-based native top-down mass spectrometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Native top-down mass spectrometry is a powerful tool for protein assembly investigation.
• Analyzing larger and more heterogeneous biomolecular assemblies requires advancements in mass analyzers and fragmentation techniques.

Purpose of the Study:

• To expand the capabilities of native top-down mass spectrometry for large and heterogeneous macromolecular complexes.
• To explore the application of ultraviolet photodissociation (UVPD) on an Orbitrap mass analyzer for analyzing complexes exceeding 1 million Da.

Main Methods:

• Utilized ultraviolet photodissociation (UVPD) on an Orbitrap mass analyzer with an extended mass range.
• Applied UVPD to large and heterogeneous biomolecular assemblies, including protein complexes and virus-like particles.
• Simultaneously monitored precursor ions, ejected subunits, and subunit fragments.

Main Results:

• Demonstrated UVPD's ability to eject intact subunits and induce backbone fragmentation for subunit identification in complexes >1 million Da.
• Successfully analyzed the light harvesting complex B-phycoerythrin, a CRISPR-Cas Csy complex, and a virus-like proteinaceous nano-container.
• Obtained partial sequence coverage, revealing chromophore localization, backbone fragmentation patterns, and sequence modifications.

Conclusions:

• UVPD-based native top-down mass spectrometry is feasible for analyzing large and heterogeneous particles, including ribonucleoprotein and megadalton virus-like particles.
• This technique provides valuable structural and sequence information for complex biomolecular assemblies.
• Advances in mass analyzers and fragmentation techniques are crucial for extending the scope of native top-down mass spectrometry.