# Next-generation Mass Spectrometry Technologies for Integrated Structural Proteomics- Renewal

> **NIH NIH R01** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2024 · $541,257

## Abstract

Project Summary
Within each organism proteins are at work carrying out activities which impact every aspect of cellular function.
A key factor in achieving such a wide range of protein functions involves the post translational modifications
and sequence modifications that act to produce a vast array of functional protein states form a single gene
product. Such proteoforms are further coupled directly to the 3D structures of the biomolecules created, which
are further recruited into a wide array of dynamic multi-protein machines. Directly assessing the structures of
these assembly states, along with the proteoforms that they contain is crucial for understanding human
disease. Despite this, most structures remain unknown and are refractory to current technologies, and their
proteoform compliment remains opaque. Standard structural biology approaches (X-ray, NMR, and Cryo-EM),
while highly successful, require pure samples in large quantities, painstakingly optimized to produce
monodisperse protein populations in every respect, and to remove spectral background. Furthermore, transient
and polydisperse assemblies that exist within complex mixtures cannot be analyzed. Mass spectrometry (MS)
approaches developed to attack this challenging problem can overcome many of these obstacles. While these
tools are undergoing a rapid development phase, they currently lack the ability to discreetly assess the
influence of proteoforms on multiprotein organization. Consequently there is a need to develop improved MS
approaches capable of simultaneously assessing the structural proteome, enabling links between proteoform
composition and 3D structure for the host of dynamic, heterogeneous macromolecular complexes of clear
biomedical importance.
This renewal application seeks to construct new, innovative MS techniques that 1) utilize new classes of
chemical tagging reagents and mixed tagging methodologies to promote comprehensive sequencing of intact
multi-protein complexes, 2) leverage next-generation cyclic ion mobility-mass spectrometry (IM-MS)
technology to produce high-definition collision induced unfolding (CIU) and native top-down sequencing
methods that enable improved identification of proteoforms within assemblies, 3) produce new techniques for
the direct sequencing of membrane protein complexes, including laser-based activation of detergent clusters
for improved data quality, 4) combine electron capture dissociation (ECD) with CIU for on-the-fly annotation of
fingerprint data, and 5) automated methods for protein derivatization and clean-up compatible with native
proteomics. This technology will be brought to bear to discover the structures of a series of selected
proteoforms and complexes, each linked to human disease.

## Key facts

- **NIH application ID:** 10913458
- **Project number:** 5R01GM095832-12
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Brandon T Ruotolo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $541,257
- **Award type:** 5
- **Project period:** 2011-04-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10913458

## Citation

> US National Institutes of Health, RePORTER application 10913458, Next-generation Mass Spectrometry Technologies for Integrated Structural Proteomics- Renewal (5R01GM095832-12). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10913458. Licensed CC0.

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