# Hybrid SID, IM, and UVPD Methods for Complex-Down MS of Protein Complexes

> **NIH NIH P41** · OHIO STATE UNIVERSITY · 2020 · $169,238

## Abstract

In order to fulfill their functions many protein complexes must first form dynamic complexes with multiple other
proteins or binding partners. Characterization of the overall stoichiometry, topology, and inter- and intra-subunit
contacts of protein and nucleoprotein complexes, and their assembly/disassembly, is critical because these
complexes regulate key biological processes. Native mass spectrometry (nMS), particularly in combination with
ion mobility (IM), and activation methods such as collision-induced dissociation (CID), ultraviolet
photodissociation (UVPD), and surface induced dissociation (SID), is emerging as a powerful technique with
which to study these complex systems and for guiding appropriate application of other structural biology tools.
Despite the promise of nMS for structural biology, commercial instruments lack many of the tools necessary to
fully characterize these complexes. SID, which is not yet commercialized, has proven to be an incredibly useful
tool in the study of protein complexes, cleaving the weakest interfaces in the complex and producing sub-
complexes that are reflective of the structure’s connectivity. While IM is commercially available on some
platforms, the resolution is often insufficient for detailed structural studies. In TR&D1, we propose to enable
higher energy SID to be performed, with more efficient fragment ion collection on multiple different instrument
platforms. In TR&D2 we propose to develop high-resolution IM on a high-resolution Orbitrap instrument. In this
TR&D we propose to couple the technologies developed in TR&Ds1 and 2, in addition to vendor prototype IM
devices, in order to enable the full characterization of protein complexes using integrated, efficient workflows.
Coupling of SID and IM is essential because when SID is placed before IM it allows conformational information
to be obtained on the intact complex and the subcomplexes produced from SID even when the peaks overlap in
m/z space, enabling structural models to be built. When SID is placed after IM, it allows different conformations
of the intact complex (if present) to be individually mobility-selected for fragmentation. In addition to coupling SID
and IM, we propose to combine these approaches with UVPD. This allows for the interrogation of complex
assembly and subunit connectivity with SID and IM, with covalent fragmentation (sequencing of the peptide
backbone) from UVPD. This approach will be beneficial in discerning ligand binding sites along with the sites of
any post-translational modifications (PTMs). We propose to do this on multiple instrumental platforms, including
the Waters Synapt G2(S), Thermo (Q) Exactive, and Bruker FTICR. The use of multiple platforms is necessary
as each platform has different mass resolution, sensitivity, and speed, and certain platforms will be better suited
to certain complexes. Hence incorporation with multiple platforms allows the experiments to be customized to
the complex of interest. The u...

## Key facts

- **NIH application ID:** 9978848
- **Project number:** 5P41GM128577-03
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** Joshua David Gilbert
- **Activity code:** P41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $169,238
- **Award type:** 5
- **Project period:** — → —

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9978848

## Citation

> US National Institutes of Health, RePORTER application 9978848, Hybrid SID, IM, and UVPD Methods for Complex-Down MS of Protein Complexes (5P41GM128577-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9978848. Licensed CC0.

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