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Title: Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster

Abstract

We present that quantitative analysis of proteomes across multiple time points, organelles, and perturbations is essential for understanding both fundamental biology and disease states. The development of isobaric tags (e.g. TMT) have enabled the simultaneous measurement of peptide abundances across several different conditions. These multiplexed approaches are promising in principle because of advantages in throughput and measurement quality. However, in practice existing multiplexing approaches suffer from key limitations. In its simple implementation (TMT-MS2), measurements are distorted by chemical noise leading to poor measurement accuracy. The current state-of-the-art (TMT-MS3) addresses this, but requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter ion approach (TMTc) produces high accuracy measurements and is compatible with many more instruments, like quadrupole-Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2-isolation step into the deconvolution algorithm. The resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty cycle, and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative label-free proteomics of total HeLamore » lysate and find that TMTc+ quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time the median coefficient of variation improves from 13% to 4%. Furthermore, TMTc+ advances quantitative proteomics by enabling accurate, sensitive, and precise multiplexed experiments on more commonly used instruments.« less

Authors:
 [1];  [1]; ORCiD logo [1]
  1. Princeton Univ., NJ (United States). Department of Molecular Biology and the Lewis-Sigler Institute for Integrative Genomics
Publication Date:
Research Org.:
Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1436582
Grant/Contract Number:  
SC0018420
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Analytical Chemistry
Additional Journal Information:
Journal Volume: 90; Journal Issue: 8; Journal ID: ISSN 0003-2700
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Sonnett, Matthew, Yeung, Eyan, and Wuhr, Martin. Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster. United States: N. p., 2018. Web. doi:10.1021/acs.analchem.7b04713.
Sonnett, Matthew, Yeung, Eyan, & Wuhr, Martin. Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster. United States. doi:10.1021/acs.analchem.7b04713.
Sonnett, Matthew, Yeung, Eyan, and Wuhr, Martin. Fri . "Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster". United States. doi:10.1021/acs.analchem.7b04713.
@article{osti_1436582,
title = {Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster},
author = {Sonnett, Matthew and Yeung, Eyan and Wuhr, Martin},
abstractNote = {We present that quantitative analysis of proteomes across multiple time points, organelles, and perturbations is essential for understanding both fundamental biology and disease states. The development of isobaric tags (e.g. TMT) have enabled the simultaneous measurement of peptide abundances across several different conditions. These multiplexed approaches are promising in principle because of advantages in throughput and measurement quality. However, in practice existing multiplexing approaches suffer from key limitations. In its simple implementation (TMT-MS2), measurements are distorted by chemical noise leading to poor measurement accuracy. The current state-of-the-art (TMT-MS3) addresses this, but requires specialized quadrupole-iontrap-Orbitrap instrumentation. The complement reporter ion approach (TMTc) produces high accuracy measurements and is compatible with many more instruments, like quadrupole-Orbitraps. However, the required deconvolution of the TMTc cluster leads to poor measurement precision. Here, we introduce TMTc+, which adds the modeling of the MS2-isolation step into the deconvolution algorithm. The resulting measurements are comparable in precision to TMT-MS3/MS2. The improved duty cycle, and lower filtering requirements make TMTc+ more sensitive than TMT-MS3 and comparable with TMT-MS2. At the same time, unlike TMT-MS2, TMTc+ is exquisitely able to distinguish signal from chemical noise even outperforming TMT-MS3. Lastly, we compare TMTc+ to quantitative label-free proteomics of total HeLa lysate and find that TMTc+ quantifies 7.8k versus 3.9k proteins in a 5-plex sample. At the same time the median coefficient of variation improves from 13% to 4%. Furthermore, TMTc+ advances quantitative proteomics by enabling accurate, sensitive, and precise multiplexed experiments on more commonly used instruments.},
doi = {10.1021/acs.analchem.7b04713},
journal = {Analytical Chemistry},
number = 8,
volume = 90,
place = {United States},
year = {Fri Mar 09 00:00:00 EST 2018},
month = {Fri Mar 09 00:00:00 EST 2018}
}

Journal Article:
Free Publicly Available Full Text
This content will become publicly available on March 9, 2019
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