# Novel NeuCode Tagging Reagents for Identification and Quantification of Intact Proteoforms in Cancer Tissues

> **NIH NIH R21** · UNIVERSITY OF WISCONSIN-MADISON · 2020 · $184,890

## Abstract

Project Summary/Abstract
In this proposal we address a very substantial limitation in the ability of existing technologies to
provide critical proteomic information from normal and cancer tissues. In standard proteomic
analyses, proteins in complex protein mixtures such as total cell lysate are digested with
proteases such as trypsin to produce peptides. These peptides are then analyzed by liquid
chromatography and mass spectrometry (LC-MS), and the proteins present are identified by the
presence of peptides contained within them. Relative or absolute quantification may be obtained
if desired by a variety of different isotopic tagging strategies. While this strategy, termed
“bottom-up” proteomics, works well for identifying large numbers of proteins present in complex
samples, it suffers from the loss of contextual information about the particular form of the
proteins from which the peptides are derived, the “proteoforms”. In order to understand the
processes and pathways that are operative in cancer, it is essential to know the identities and
abundances of these “proteoforms” present in the tissues.
Recently, a new approach has been developed for the identification and quantification of
proteoforms in complex mixtures. In this approach two pieces of information are obtained for
each proteoform in the sample: a highly accurate measurement of the proteoform intact mass,
and the number of lysine residues it contains. This information allows identification and
quantification of thousands of proteoforms. However, at present it can only be performed on
cells grown in culture containing isotopically labeled lysine amino acids. This limitation makes it
impossible to use the strategy for identification of proteoforms in cancer tissue samples. It is
proposed here to develop an alternative means of introducing the isotopic tags needed for
proteoform quantification and for the determination of the number of a targeted amino acid
residue in each proteoform. Cysteine amino acids have been chosen in place of lysine amino
acids because of their amenability to highly efficient tagging reactions, and suitable isotopic
labels have been designed and will be synthesized and tested. This new chemistry will provide
the power of the isotopic tagging strategy for proteomic analyses of cancer tissue samples.
Such proteoform level knowledge of changes that occur in cancer will reveal a new universe of
possible cancer biomarkers, as well as opening a currently closed avenue to understanding the
proteomic changes that occur in cancer.

## Key facts

- **NIH application ID:** 9880284
- **Project number:** 5R21CA223887-03
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** LLOYD M SMITH
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $184,890
- **Award type:** 5
- **Project period:** 2018-03-14 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9880284

## Citation

> US National Institutes of Health, RePORTER application 9880284, Novel NeuCode Tagging Reagents for Identification and Quantification of Intact Proteoforms in Cancer Tissues (5R21CA223887-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9880284. Licensed CC0.

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