Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics

Research output: Contribution to journalArticlepeer-review

Standard

Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics. / Pinto-Fernández, Adán; Davis, Simon; Schofield, Abigail B; Scott, Hannah C; Zhang, Ping; Salah, Eidarus; Mathea, Sebastian; Charles, Philip D; Damianou, Andreas; Bond, Gareth; Fischer, Roman; Kessler, Benedikt M.

In: Frontiers in Chemistry, Vol. 7, 592, 29.08.2019, p. 1-14.

Research output: Contribution to journalArticlepeer-review

Harvard

Pinto-Fernández, A, Davis, S, Schofield, AB, Scott, HC, Zhang, P, Salah, E, Mathea, S, Charles, PD, Damianou, A, Bond, G, Fischer, R & Kessler, BM 2019, 'Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics', Frontiers in Chemistry, vol. 7, 592, pp. 1-14. https://doi.org/10.3389/fchem.2019.00592

APA

Pinto-Fernández, A., Davis, S., Schofield, A. B., Scott, H. C., Zhang, P., Salah, E., Mathea, S., Charles, P. D., Damianou, A., Bond, G., Fischer, R., & Kessler, B. M. (2019). Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics. Frontiers in Chemistry, 7, 1-14. [592]. https://doi.org/10.3389/fchem.2019.00592

Vancouver

Pinto-Fernández A, Davis S, Schofield AB, Scott HC, Zhang P, Salah E et al. Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics. Frontiers in Chemistry. 2019 Aug 29;7:1-14. 592. https://doi.org/10.3389/fchem.2019.00592

Author

Pinto-Fernández, Adán ; Davis, Simon ; Schofield, Abigail B ; Scott, Hannah C ; Zhang, Ping ; Salah, Eidarus ; Mathea, Sebastian ; Charles, Philip D ; Damianou, Andreas ; Bond, Gareth ; Fischer, Roman ; Kessler, Benedikt M. / Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics. In: Frontiers in Chemistry. 2019 ; Vol. 7. pp. 1-14.

Bibtex

@article{dec930eff38c4fbebecd42dc000569e1,
title = "Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics",
abstract = "Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.",
author = "Ad{\'a}n Pinto-Fern{\'a}ndez and Simon Davis and Schofield, {Abigail B} and Scott, {Hannah C} and Ping Zhang and Eidarus Salah and Sebastian Mathea and Charles, {Philip D} and Andreas Damianou and Gareth Bond and Roman Fischer and Kessler, {Benedikt M}",
year = "2019",
month = aug,
day = "29",
doi = "10.3389/fchem.2019.00592",
language = "English",
volume = "7",
pages = "1--14",
journal = "Frontiers in Chemistry",
issn = "2296-2646",
publisher = "Frontiers Media S.A.",

}

RIS

TY - JOUR

T1 - Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics

AU - Pinto-Fernández, Adán

AU - Davis, Simon

AU - Schofield, Abigail B

AU - Scott, Hannah C

AU - Zhang, Ping

AU - Salah, Eidarus

AU - Mathea, Sebastian

AU - Charles, Philip D

AU - Damianou, Andreas

AU - Bond, Gareth

AU - Fischer, Roman

AU - Kessler, Benedikt M

PY - 2019/8/29

Y1 - 2019/8/29

N2 - Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.

AB - Enzymes that bind and process ubiquitin, a small 76-amino-acid protein, have been recognized as pharmacological targets in oncology, immunological disorders, and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition to the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation, and more potent/selective ubiquitin active-site probes with propargylic-based electrophiles to profile 74 DUBs including distinguishable isoforms for 5 DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents and pan-DUB inhibitors combined with probe labeling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39, and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small-molecule DUB selectivity profiling.

U2 - 10.3389/fchem.2019.00592

DO - 10.3389/fchem.2019.00592

M3 - Article

C2 - 31555637

VL - 7

SP - 1

EP - 14

JO - Frontiers in Chemistry

JF - Frontiers in Chemistry

SN - 2296-2646

M1 - 592

ER -