Uncovering hidden protein modifications with native top-down mass spectrometry

Area of Science:

• Proteomics
• Structural Biology
• Biochemistry

Background:

• Protein modifications are crucial for cellular functions but challenging to study in their native context.
• Native top-down mass spectrometry offers a way to link modifications with interactions.
• Current methods struggle to detect low-abundance or uncharacterized modifications, limiting proteoform diversity insights.

Purpose of the Study:

• To introduce precisION, a software package for detecting and localizing 'hidden' protein modifications within intact complexes.
• To enable accurate quantification of these modifications using native top-down mass spectrometry.
• To advance the study of proteoform diversity and its implications.

Main Methods:

• Development of precisION, an interactive end-to-end software package.
• Utilizing a data-driven fragment-level open search strategy.
• Application to therapeutically relevant targets: PDE6, ACE2, osteopontin (SPP1), and GAT1.

Main Results:

• Discovery of previously undocumented phosphorylation, glycosylation, and lipidation events.
• Successful detection and localization of 'hidden' modifications in intact protein complexes.
• Resolution of uninterpretable density in an electron cryo-microscopy map of GAT1.

Conclusions:

• precisION is an open-source tool that facilitates the interpretation of complex protein fragmentation data.
• The software empowers researchers to leverage native top-down mass spectrometry for deeper insights.
• This advancement supports integrative structural biology, molecular pathology, and drug development.