# Advancing native top-down protein analysis with hybrid SID/ECD technology

> **NIH NIH R43** · E-MSION, INC. · 2021 · $252,070

## Abstract

The capabilities of native mass spectrometry have improved dramatically in recent years due to advances in
instrument speed, resolution, mass accuracy, and ion optics designed specifically to handle large protein
complexes. Despite these advances, the depth of characterization achievable by native mass spectrometry is
still limited due to inefficient dissociation and fragmentation of large protein complexes within the mass
spectrometer. At e-MSion, Inc., we have developed an efficient electron-fragmentation technology called ExD
now co-marketed with Agilent for their family of Q-TOFs, with Waters for their Q-IM-TOFs, and with
Thermo for their Ultra High Mass Range (UHMR) Orbitraps. The ExD technology provides extensive
fragmentation of denatured and native proteins enabling thorough sequencing and localization of
posttranslational modifications. However, the large masses of many protein complexes now accessible by
mass spectrometry make them particularly challenging to dissociate and fragment by electron-based ion
activation methods alone. Surface induced dissociation (SID) is a complementary technique capable of
dissociating large protein complexes to reveal higher order structure, such as subunit stoichiometry,
topology, and interfaces, with minimal unfolding and fragmentation of the subunits. However, SID is
incapable of separating intermolecular disulfide crosslinked proteins. We have shown our ExD technology is
extremely effective at cutting multiple disulfide bonds in Cysteine Knot Proteins and monoclonal antibodies.
Recent advances in the Wysocki lab at Ohio State University have resulted in a remarkable shortening of
their SID design, which now makes it possible to combine the two complementary approaches of native
protein dissociation and fragmentation. In this phase I proposal, we will evaluate the feasibility of developing
a hybrid ExD-SID cell for the UHMR Orbitrap mass spectrometer to characterize disulfide-crosslinked native
protein complexes. Ion optics and electronics required to perform SID will be integrated into the ExD cell
and ExD controller for the UHMR Orbitrap platform. We will optimize the hybrid cell design to maximize ion
transmission, ExD, SID, and ExD-SID experiments. The developed hybrid cell and methods will be applied
the characterization of native antibodies as a model system. The hybrid ExD-SID cell will enable efficient
fragmentation of disulfide bonds and dissociation of noncovalent interactions enabling separation of the
intact heavy and light chains of the antibody. Success in addressing the feasibility question will yield a
powerful tool for rapid characterization and discovery of monoclonal antibody therapeutics. More broadly,
successful development of a hybrid ExD-SID cell will create a tool capable of bringing native mass
spectrometry into the mainstream for structural biology approaches by greatly expanding the mass range of
macromolecular complexes amenable to extensive characterization.

## Key facts

- **NIH application ID:** 10155281
- **Project number:** 1R43GM140749-01
- **Recipient organization:** E-MSION, INC.
- **Principal Investigator:** Jared Bryan Shaw
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $252,070
- **Award type:** 1
- **Project period:** 2021-01-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10155281

## Citation

> US National Institutes of Health, RePORTER application 10155281, Advancing native top-down protein analysis with hybrid SID/ECD technology (1R43GM140749-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10155281. Licensed CC0.

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