# New and Disruptive Technologies to Study Ubiquitin Biology through Sample Multiplexing

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2020 · $508,500

## Abstract

Defects in ubiquitin (Ub) pathways are often responsible for cancer and devastating neurodegenerative
diseases. Because of its central role in biological circuits and the potential for therapeutic intervention, the Ub
system is an intense research area. Yet, Ub biology is highly complex—in humans it is supported by over 500
Ub ligases and 95 deubiquitinases. Currently, this complexity and our limited understanding of players and
their interactions are a severe hindrance for targeted intervention in many diseases. Considering all possible
ways to address the complexity, sample multiplexing in mass spectrometry-based proteomics arguably has the
greatest potential to fundamentally revolutionize the throughput of these measurements. Through efforts within
the previous grant cycle, the level of multiplexing was increased, facilitating the quantitative comparison of
expression and conjugate levels for 10 samples. In this proposal, enabling advances in sample multiplexing
will be explored and applied to study ubiquitin-dependent biology. In Aim 1, we will evaluate isobaric reagents
that have multiple reporter ions such that a 16- and a 32-plex reagent set can be synthesized using relatively
few heavy atoms (4 and 6, respectively). In Aim 2, two large obstacles to sample multiplexing will be
addressed—sensitivity and proteome depth. Using a real-time database search algorithm, reporter ion
quantification scans (SPS-MS3 scans) will only be collected when the MS2 scan is successfully matched to a
peptide or PTM-containing peptide. This will be a very disruptive technology as MS2 scans are recorded at a
blistering rate of 22 Hz. Highly optimized MS3 scans will then be collected based on any filter desired since
the identity and modification state of all peptides will be known in real time. We expect dramatic improvements
in the quality and depth of discovery proteomics experiments while reducing the analysis time by at least 3 fold.
 In Aim 3, we will explore two new multiplexing workflows based on the technologies developed in the
first two aims. One workflow will be dedicated to targeted proteomics where up to 32-plex experiments
targeting hundreds of peptides will be developed. In these assays, triggering peptides and a real-time
database search will obviate the need for retention time scheduling or precursor detection in survey scans prior
to MS3 analysis using pre-selected SPS ions. Using panels of trigger peptides, assays to monitor the protein
levels in six ubiquitin-dependent pathways will be created. The second workflow will be dedicated to discovery
proteomics where up to 32-plex experiments will be conducted for full proteome or conjugate analysis in as
little as 24 hr. This will be applied to characterize CRISPR deletions in the mTOR amino acid sensing pathway
and specifically the mechanism of action of the GATOR2 complex where 3 ubiquitin ligases of unknown
function are present. The realization of all three aims ushers in a paradigm shift in Qu...

## Key facts

- **NIH application ID:** 9873038
- **Project number:** 5R01GM067945-17
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** STEVEN P GYGI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $508,500
- **Award type:** 5
- **Project period:** 2003-05-15 → 2021-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9873038

## Citation

> US National Institutes of Health, RePORTER application 9873038, New and Disruptive Technologies to Study Ubiquitin Biology through Sample Multiplexing (5R01GM067945-17). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9873038. Licensed CC0.

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