# Chemistry for next-generation single-molecule fluorosequencing technology 2.0.

> **NIH NIH R01** · EMORY UNIVERSITY · 2023 · $2,090,618

## Abstract

PROJECT SUMMARY
The human proteome is extremely complex, comprising > 10,000 proteins and 100 times proteoforms for each
gene product. In cancer and other diseases, several new protein variants may result from mutations, fusions
and PTMs that further influence the functions and structure of proteins. This necessitates the identification of
proteins and PTMs at a single-molecule level in a cell or an organism to understand biological processes,
disease analysis and biomarker discovery. Despite the power of protein sequencing in revolutionizing precision
medicine diagnostics, there are no single-molecule methods to identify proteins and PTMs at the proteome-
wide level. Therefore, there is a huge gap in understanding the role of proteins and PTMs in biology and
diseases due to the lack of efficient techniques for the analysis of low abundant proteins and PTMs at a single-
molecule level in a highly complex proteome system. The main goal of this research proposal is to fill the
present gap in the range of available techniques to sequence and identify proteins and PTMs at the single-
molecule level. A new suite of chemical methods will be developed for specific modification of side chains of
amino acids and PTMs that are of low reactivity thus challenging to modify, to attach various fluorescent
moieties to peptides. As a trained organic chemist and chemical biologist, and in collaboration with the
founders (Dr. Eric Anslyn and Dr. Ed Marcotte) of single-molecule protein fluorosequencing, we are positioned
to rapidly evaluate our newly developed chemical methods for the proteome-wide analyses in a high
throughput manner. A high degree of chemical specificity and yield of the new chemical methods will avoid
downstream misidentification of amino acids by single-molecule fluorosequencing. The proposed research
contains various innovations for advancing single-molecule protein sequencing. The First innovation, involves
the chemical methods for the selective labeling of methyl lysine and methyl histidine PTMs, such as
(monomethyl lysine Kme, dimethyl lysine Kme2, trimethyl lysine Kme3 and methylhistidine Hme) that are
compatible with single molecule fluorosequencing. The second innovation is the development of chemical
methods for the selective labeling of less reactive amino acids, such as amides (Gln and Asn), ethers (Met)
and alkanes (Ile, Leu, Val, Phe, Pro) that are compatible with single molecule fluorosequencing. These new
chemical methods for single molecule fluorosequencing will lead to the identification of amino acids and PTMs
with high sensitivity, accuracy, and dynamic range capable of identifying low abundant proteins and PTMs at
the proteome-wide scale in a high throughput manner. Thus, the proposed research has a great potential to
further our understanding of how these PTMs regulate various cellular signaling processes and lead to various
diseases. Such tools would lead to the discovery of novel methyl lysine and methyl histidine biomarke...

## Key facts

- **NIH application ID:** 10645898
- **Project number:** 1R01HG012941-01
- **Recipient organization:** EMORY UNIVERSITY
- **Principal Investigator:** Monika Raj
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $2,090,618
- **Award type:** 1
- **Project period:** 2023-06-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10645898

## Citation

> US National Institutes of Health, RePORTER application 10645898, Chemistry for next-generation single-molecule fluorosequencing technology 2.0. (1R01HG012941-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10645898. Licensed CC0.

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