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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]
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  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:
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.
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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.
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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. https://www.osti.gov/servlets/purl/1436582.
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@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 = {2018},
month = {3}
}
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Journal Article:
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Publisher's Version of Record
DOI:  10.1021/acs.analchem.7b04713
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Cited by: 9 works
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Figures / Tables:

Figure 1 Figure 1: Modeling the isolation step in the deconvolution algorithm increases measurement precision. A) Five different TMT reagents are used to barcode five identical samples of peptides from a HeLa lysate. An example of the isotopic envelope of an intact peptide (precursor) is shown. The true underlying ratios are shownmore » in color, but the mass spectrometer is blind to the barcoding and only what is shown in black is observed. Note that in the MS1 before any fragmentation has occurred that the true ratio of 1:1:1:1:1 is present in each peak of the envelope. B) In the published form of TMTc the entire monoisotopic envelope is first isolated and then fragmented. The peaks corresponding to the complement reporter ions are identified and the relative abundances are determined. The simultaneous fragmentation of multiple peaks from the precursor convolves the data. Intuition for this convolvement is provided in Fig S1. Using the known theoretical distribution of charge states from the precursor, the amount of signal in each mass offset that belongs to each condition can be estimated using a least- squares optimization.12 This process is done for thousands of peptides and the resulting histograms of the measured ratio are shown, resulting in a median CV of 16%. C) With TMTc+ the shape of a much narrower (e.g. 0.5 Th) isolation window (red) is measured and used. The shape and position of this window is incorporated into the deconvolution algorithm. With narrower isolation windows deconvolution becomes easier and precision is gained. In the extreme case where only a single peak from the envelope is isolated, the TMTc+ algorithm has to only calculate away isotopic impurities from the TMT-tag. The median CV on the peptide level improves to 6%. D) TMTc+ can accommodate isolation windows of any size as long as their shape has been measured. In the case where a 1.0 Th window is used (red), a small amount of the M+2 peak is isolated in addition to the M+1 peak. Using the shape of this moderately sized isolation window, the ratios can be deconvolved and a significant improvement in precision (median CV of 8%) is still observed relative to when the entire isotopic envelope is isolated with previously published TMTc.« less
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