# Revealing Proteoforms: The Primary Effectors of Biological Function

> **NIH NIH R35** · UNIVERSITY OF WISCONSIN-MADISON · 2024 · $726,212

## Abstract

ABSTRACT
The primary focus of my laboratory is the development of new tools and strategies for proteomic analyses of
complex biological systems, specifically centered around the concept of the proteoform. Proteoforms, each of
which comprises a unique combination of amino acid sequence and post-translational modifications (PTMs),
are the primary molecular effectors of cell function. Subtle sequence and PTM differences between
proteoforms can completely alter their function and activity. We see comprehensive proteoform-level analysis
of biological systems as absolutely essential to understanding their function, for both individual pathways and
networks operative within cells, and more globally, to decipher the systems-biology-level dynamics and
interactions that control cellular response. The current technology for global proteoform analysis in complex
systems is in its infancy, offering both a great challenge and a great opportunity. Our laboratory is keenly
interested in tackling this problem and is pioneering a new approach that integrates high resolution proteoform
intact mass measurements, both bottom-up and top-down strategies, new informatic tools for the
comprehensive analysis of PTMs, and RNA-Seq information; all woven together in a robust bioinformatic
framework to allow the comprehensive identification and quantification of proteoforms in complex mixtures.
Along with other world-class scientists, we will work towards embarking on the Human Proteoform Project,
which includes ambitious subprojects describing the construction and utility of comprehensive proteoform
atlases for humans and model organisms. Specific projects in our laboratory will include development of the
following: (1) a multi-dimensional separation strategy for increased breadth and depth of proteoform
identifications; (2) a source-induced dissociation method for fragmentation of eluting proteoform ions to
increase proteoform identifications; (3) intelligent real-time data acquisition; (4) direct acquisition of orbitrap
time-domain transients to expand the accessible mass range; (5) data analysis software including the abilities
to search for truncated proteoforms and utilize the most abundant mass for identification; (6) sample-specific
databases created through integration of bottom-up, top-down, intact mass and RNA-Seq data; (7)
visualization tools for manual validation of proteoform identifications and for troubleshooting problems with
samples and/or algorithms; and (8) using proteoform quantitative trait loci (QTLs) to reveal the modifying
enzymes encoded elsewhere in the genome that are responsible for the critical post-translational modifications
with functional consequence. We are excited to develop powerful new tools to advance the state-of-the-art in
this new and important field of study to reveal the biologically important effectors of cellular mechanisms.
These tools, which will be made widely available to all researchers, will reveal new information essential ...

## Key facts

- **NIH application ID:** 10934319
- **Project number:** 5R35GM126914-07
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** LLOYD M SMITH
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $726,212
- **Award type:** 5
- **Project period:** 2018-05-01 → 2028-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10934319

## Citation

> US National Institutes of Health, RePORTER application 10934319, Revealing Proteoforms: The Primary Effectors of Biological Function (5R35GM126914-07). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10934319. Licensed CC0.

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