# Protein Sequencing by Tandem Mass Spectrometry

> **NIH NIH R01** · UNIVERSITY OF VIRGINIA · 2021 · $757,113

## Abstract

Project Summary
Proposed here is research to develop a collection of technologies that will make it possible to read the complete
amino acid sequence of proteins (forwards and backwards) on a chromatographic time scale at the femtomole
level and to also characterize multiple posttranslational modifications that exist on the same protein molecule
and together regulate its biological activity. This research is driven by four major innovations from my lab. These
include development of (a) electron transfer dissociation (ETD) for fragmentation of peptides and intact proteins,
(b) combined ETD and IIPT (ion-ion proton transfer) chemistry to obtain n- and c-terminal sequence information
that identifies proteins, (c) front end ETD (FETD) that facilitates a 10-50 fold increase in sensitivity for intact
proteins, and (d) a micro column enzyme reactor that is fully active in 8 M urea and can be employed to generate
3-10 KDa protein fragments from large proteins. Going forward, we will implement and optimize ETD and IIPT
in combination with parallel ion parking (PIP). This will include the introduction of new ETD reagents and new
wave forms to enhance the efficiency of parallel ion parking and new IIPT reagents to extend the usable mass
range to m/z 4,000. Also proposed is research to incorporate the powerful micro-column enzyme reactor into a
rotation-driven microfluidic disc system where the protease bed length, solvent flow in hundreds of nL/min, and
digestion times on the order of hundreds of milliseconds can all be controlled and measured with high accuracy.
By using the rotation-driven microfluidic disc system, we expect to increase sample throughput, lower sample
quantities, and increase reproducibility of individual sample analyses. All of this will be applied to fully
characterize (a) bispecific antibodies, (b) proteins in outer membrane vesicles secreted by the antibiotic resistant,
gram negative bacteria, Neisseria gonorrhoeae, (c) plant proteins that are involved in growth or growth inhibition
and are targets for O-GlcNAcylation and O-fucosylation, respectively, (d) posttranslational modifications that
exist on histone chaperones during the cell cycle, and (e) posttranslational modifications that regulate
methylation of Arg residues on histones H3 and H4.

## Key facts

- **NIH application ID:** 10171864
- **Project number:** 5R01GM037537-34
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** DONALD F HUNT
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $757,113
- **Award type:** 5
- **Project period:** 1987-01-12 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10171864

## Citation

> US National Institutes of Health, RePORTER application 10171864, Protein Sequencing by Tandem Mass Spectrometry (5R01GM037537-34). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10171864. Licensed CC0.

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