Uncovering hidden protein modifications with native top-down mass spectrometry

Journal article

Protein modifications drive dynamic cellular processes by modulating biomolecular

interactions, yet capturing these modifications within their native structural context remains a

significant challenge. Native top-down mass spectrometry (nTDMS) promises to preserve the

critical link between modifications and interactions. However, current methods often fail to

detect uncharacterized or low-abundance modifications, limiting insights into proteoform

diversity. To address this gap, we introduce precisION, an interactive end-to-end software

package that leverages a robust, data-driven fragment-level open search to detect, localize,

and quantify “hidden” modifications within intact protein complexes. Applying precisION to

four therapeutically relevant targets—PDE6, ACE2, osteopontin (SPP1), and a GABA

transporter (GAT1)—here we discover undocumented phosphorylation, glycosylation, and

lipidation, and resolve previously uninterpretable density in an electron cryo-microscopy map

of GAT1. As an open-source software package, precisION offers an intuitive means for

interpreting complex protein fragmentation data. This tool will empower the community to

unlock the potential of nTDMS, advancing integrative structural biology, molecular pathology,

and drug development.

Authors: Bennett, J; El Baba, T; Zouboulis, KC; Kirschbaum, C; Song, H

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Springer Nature
• Journal: Nature Methods
• Volume: 22
• Issue: 10
• Pages: 2127–2137
• Publication date: 2025-09-29
• Acceptance date: 2025-08-21
• DOI: 10.1038/s41592-025-02846-5
• EISSN: 1548-7105
• ISSN: 1548-7091
• Language: English
• Keywords: structural biology; proteins; post-translational modifications; native mass spectrometry; top-down mass spectrometry; proteoform