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Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes

[Image: see text] The study of low-abundance proteins is a challenge to discovery-based proteomics. Mass spectrometry (MS) applications, such as thermal proteome profiling (TPP), face specific challenges in the detection of the whole proteome as a consequence of the use of nondenaturing extraction b...

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Autores principales: Peck Justice, Sarah A., McCracken, Neil A., Victorino, José F., Qi, Guihong D., Wijeratne, Aruna B., Mosley, Amber L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2021
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153406/
https://www.ncbi.nlm.nih.gov/pubmed/33908254
http://dx.doi.org/10.1021/acs.analchem.1c00012
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author Peck Justice, Sarah A.
McCracken, Neil A.
Victorino, José F.
Qi, Guihong D.
Wijeratne, Aruna B.
Mosley, Amber L.
author_facet Peck Justice, Sarah A.
McCracken, Neil A.
Victorino, José F.
Qi, Guihong D.
Wijeratne, Aruna B.
Mosley, Amber L.
author_sort Peck Justice, Sarah A.
collection PubMed
description [Image: see text] The study of low-abundance proteins is a challenge to discovery-based proteomics. Mass spectrometry (MS) applications, such as thermal proteome profiling (TPP), face specific challenges in the detection of the whole proteome as a consequence of the use of nondenaturing extraction buffers. TPP is a powerful method for the study of protein thermal stability, but quantitative accuracy is highly dependent on consistent detection. Therefore, TPP can be limited in its amenability to study low-abundance proteins that tend to have stochastic or poor detection by MS. To address this challenge, we incorporated an affinity-purified protein complex sample at submolar concentrations as an isobaric trigger channel into a mutant TPP (mTPP) workflow to provide reproducible detection and quantitation of the low-abundance subunits of the cleavage and polyadenylation factor (CPF) complex. The inclusion of an isobaric protein complex trigger channel increased detection an average of 40× for previously detected subunits and facilitated detection of CPF subunits that were previously below the limit of detection. Importantly, these gains in CPF detection did not cause large changes in melt temperature (T(m)) calculations for other unrelated proteins in the samples, with a high positive correlation between T(m) estimates in samples with and without isobaric trigger channel addition. Overall, the incorporation of an affinity-purified protein complex as an isobaric trigger channel within a tandem mass tag (TMT) multiplex for mTPP experiments is an effective and reproducible way to gather thermal profiling data on proteins that are not readily detected using the original TPP or mTPP protocols.
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spelling pubmed-81534062021-05-27 Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes Peck Justice, Sarah A. McCracken, Neil A. Victorino, José F. Qi, Guihong D. Wijeratne, Aruna B. Mosley, Amber L. Anal Chem [Image: see text] The study of low-abundance proteins is a challenge to discovery-based proteomics. Mass spectrometry (MS) applications, such as thermal proteome profiling (TPP), face specific challenges in the detection of the whole proteome as a consequence of the use of nondenaturing extraction buffers. TPP is a powerful method for the study of protein thermal stability, but quantitative accuracy is highly dependent on consistent detection. Therefore, TPP can be limited in its amenability to study low-abundance proteins that tend to have stochastic or poor detection by MS. To address this challenge, we incorporated an affinity-purified protein complex sample at submolar concentrations as an isobaric trigger channel into a mutant TPP (mTPP) workflow to provide reproducible detection and quantitation of the low-abundance subunits of the cleavage and polyadenylation factor (CPF) complex. The inclusion of an isobaric protein complex trigger channel increased detection an average of 40× for previously detected subunits and facilitated detection of CPF subunits that were previously below the limit of detection. Importantly, these gains in CPF detection did not cause large changes in melt temperature (T(m)) calculations for other unrelated proteins in the samples, with a high positive correlation between T(m) estimates in samples with and without isobaric trigger channel addition. Overall, the incorporation of an affinity-purified protein complex as an isobaric trigger channel within a tandem mass tag (TMT) multiplex for mTPP experiments is an effective and reproducible way to gather thermal profiling data on proteins that are not readily detected using the original TPP or mTPP protocols. American Chemical Society 2021-04-28 2021-05-11 /pmc/articles/PMC8153406/ /pubmed/33908254 http://dx.doi.org/10.1021/acs.analchem.1c00012 Text en © 2021 The Authors. Published by American Chemical Society Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Peck Justice, Sarah A.
McCracken, Neil A.
Victorino, José F.
Qi, Guihong D.
Wijeratne, Aruna B.
Mosley, Amber L.
Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes
title Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes
title_full Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes
title_fullStr Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes
title_full_unstemmed Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes
title_short Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes
title_sort boosting detection of low-abundance proteins in thermal proteome profiling experiments by addition of an isobaric trigger channel to tmt multiplexes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153406/
https://www.ncbi.nlm.nih.gov/pubmed/33908254
http://dx.doi.org/10.1021/acs.analchem.1c00012
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