# Development of Multidimensional, Linear and Differential Ion Mobility MS Separations for Middle-Down Proteoforms

> **NIH NIH R01** · FLORIDA INTERNATIONAL UNIVERSITY · 2020 · $363,379

## Abstract

PROJECT SUMMARY/ABSTRACT
With proteomics entering the third decade, the identification and quantification of primary protein
sequences has become a mature science. However, many analytical challenges remain during the mapping
of post-translational modifications (PTMs) of proteins, especially in their native state. For example,
complete deciphering of the "histone code" entails the identification and localization of the different
PTMs using top down strategies. While some advances have been made, traditional LC-MS/MS methods
cannot fully separate histones with isomeric PTM position variants; this leads to the need to further
develop new, fast, and orthogonal separations that can be easily integrated with mass spectrometry for the
characterization of the “histone code”. In the present project, we aim to test the hypothesis that “PTMs
induce structural changes in histones, allowing their separation and identification by the difference in ion
mobility properties (particularly, for positional isomers), and fragmentation patterns”. Understanding the
effect of PTMs on histone structure and function is central to the epigenetic regulation. Thus, to enable
further advances in epigenetics, we will develop new multidimensional ion mobility separations for top-
down isoform histone analyses. The ultimate goal of this project is to bring together technology experts in
post-ionization, orthogonal separations and top-down mass spectrometry to develop an integrative,
multidimensional analytical platform, capable of characterizing the “histone code” using native, as
opposed to proteolytically digested, histones. To accomplish this goal, we will pursue the following aims:
1) To integrate multi-stage, non-linear, and linear ion mobility separations with top-down mass
spectrometry (IMSn-MS/MS); 2) To develop liquid chromatography (offline and online) strategies
compatible with IMSn-MS/MS.; and 3) To evaluate histone PTM abundances in biological systems using
LC-MS/MS and LC-IMSn-MS/MS strategies. To support these aims, major technological breakthroughs
in each of the integrated areas will be achieved: i) non-linear IMS (with 3 new FAIMS geometries
implemented and evaluated), ii) linear IMS (with a higher resolution, larger mobility range, and higher
sensitivity TIMS cell implemented and evaluated), and iii) non-ergodic top-down fragmentation (i.e., ExD
and UVPD) compatible with online, native LC and mobility workflows. Completing the aims of this
proposal will provide new innovative and enabling analytical solutions and instrumentation, that will
benefit the proteomics community at large and open new doors for functional top-down proteomics.

## Key facts

- **NIH application ID:** 10019582
- **Project number:** 5R01GM134247-02
- **Recipient organization:** FLORIDA INTERNATIONAL UNIVERSITY
- **Principal Investigator:** Francisco Fernandez-Lima
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $363,379
- **Award type:** 5
- **Project period:** 2019-09-20 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10019582

## Citation

> US National Institutes of Health, RePORTER application 10019582, Development of Multidimensional, Linear and Differential Ion Mobility MS Separations for Middle-Down Proteoforms (5R01GM134247-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10019582. Licensed CC0.

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