# Advancing Multiplexed Isobaric Tag-based Strategies for Proteome Profiling

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2020 · $339,000

## Abstract

ABSTRACT
Sample multiplexing has been the catalyst for many recent large-scale proteomics initiatives. The advent of
isobaric tagging, as popularized by iTRAQ (isobaric tags for relative and absolute quantitation) and TMT
(tandem mass tag) reagents, has become the quintessential methodology for multiplexed protein expression
profiling. Two major data acquisition methods exist each with its own advantages and disadvantages. First, the
MS2-only method (“MS2-IDQ” herein) can identify (ID) and quantify (Q) a peptide in a single spectrum.
Second, the synchronous precursor selection (SPS)-MS3 method identifies the precursor in the MS2 stage, but
then selects a series of fragment ions from the MS2 stage that are fragmented further and read out as an MS3
spectrum for quantification measurements. MS2-IDQ suffers from the co-isolation and co-fragmentation of
precursor ions (“interference”), and although SPS-MS3 helps to alleviate interference, it is at the expense of
speed, a direct result from the acquisition of long MS3 scans. Here we aim to develop, evaluate, and apply a
novel data acquisition platform that merges the benefits of current methods and alleviates their major caveats.
A recent development on ThermoFisher Scientific's Orbitrap Fusion and Lumos instruments has been the
implementation of an instrument application programming interface (iAPI) that allows for expanded control of
the instrumentation beyond the manufacturer's built-in functionality. Using this interface, the Gygi Lab and
others have begun to create custom on-the-fly real-time search (RTS) algorithms. RTS enables an MS2
spectrum to be searched in real-time and decisions to be made as to whether an MS3 scan is likely to result in
a significant peptide quantification measurement. By omitting MS3 scans, more MS2 spectra can be collected
and new peptides may be identified. Using the iAPI, functions can be added including targeted lists and limits
set for the number of peptides quantified per protein (in the case of very abundant and/or large proteins), which
is useful in translational research, such as the interrogation of plasma samples and other body fluids.
Our Specific Aims are geared toward developing further the methodology for successful application of RTS-
MS3. In Specific Aim 1, we will benchmark emerging algorithms for RTS-MS3 using both the TKO and
HYPER (human-yeast peptide resource) standards for TMT-based proteome profiling. In Specific Aim 2, we
will evaluate the RTS-MS3 platform across several sample types (bacterial cultures, mouse tissues, blood,
cerebral spinal fluid, human cell lines, and yeast cultures) against traditional MS2-IDQ and SPS-MS3 methods
(Specific Aim 2). Finally, in Specific Aim 3 we will apply the RTS-MS3 platform to analyze an entire Yeast
Deletion Strain Collection under two growth conditions, which will produce the largest yeast protein expression
profiling data set to date. Accomplishing these three Specific Aims will establish the RTS-MS3 platfo...

## Key facts

- **NIH application ID:** 10018062
- **Project number:** 5R01GM132129-02
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Joao A Paulo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $339,000
- **Award type:** 5
- **Project period:** 2019-09-15 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10018062

## Citation

> US National Institutes of Health, RePORTER application 10018062, Advancing Multiplexed Isobaric Tag-based Strategies for Proteome Profiling (5R01GM132129-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10018062. Licensed CC0.

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