Sequential digestion with Trypsin and Elastase in cross-linking mass spectrometry

Therese Dau; Kapil Gupta; Imre Berger; Juri Rappsilber

doi:10.1021/acs.analchem.8b05222

Sequential digestion with Trypsin and Elastase in cross-linking mass spectrometry

Therese Dau, Kapil Gupta, Imre Berger, Juri Rappsilber

School of Biological Sciences

Research output: Contribution to journal › Article › peer-review

Abstract

Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

Original language	English
Pages (from-to)	4472-4478
Number of pages	7
Journal	Analytical Chemistry
Volume	91
Early online date	28 Feb 2019
DOIs	https://doi.org/10.1021/acs.analchem.8b05222
Publication status	Published - 2 Apr 2019

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10.1021/acs.analchem.8b05222Licence: Creative Commons: Attribution (CC-BY)

Sequential Digestion PUB OA CC BY acs.analchem.8b05222
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Final published version, 2.25 MBLicence: Creative Commons: Attribution (CC-BY)

Wellcome Centre for Cell Biology
Tollervey, D. (Principal Investigator)
UK-based charities
1/12/16 → 1/12/21
Project: Research
Proteomics at the Wellcome Trust Centre for Cell Biology (WTCCB) and School of Biological Sciences (SBS), Edinburgh
Rappsilber, J. (Principal Investigator)
UK-based charities
1/10/15 → 30/09/20
Project: Research
Protein structures in the context of time and space by mass spectrometry
Rappsilber, J. (Principal Investigator)
UK-based charities
1/06/14 → 31/05/21
Project: Research

Sequential digestion with Trypsin and Elastase in cross-linking mass spectrometry
Dau, T. (Creator), Gupta, K. (Creator), Berger, I. (Creator) & Rappsilber, J. (Creator), PRIDE database hosted by European Bioinformatics Institute, EBI, 7 Mar 2019
Dataset

Cite this

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abstract = "Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.",

author = "Therese Dau and Kapil Gupta and Imre Berger and Juri Rappsilber",

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N2 - Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

AB - Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

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DO - 10.1021/acs.analchem.8b05222

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VL - 91

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EP - 4478

JO - Analytical Chemistry

JF - Analytical Chemistry

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Sequential digestion with Trypsin and Elastase in cross-linking mass spectrometry

Abstract

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Projects

Wellcome Centre for Cell Biology

Proteomics at the Wellcome Trust Centre for Cell Biology (WTCCB) and School of Biological Sciences (SBS), Edinburgh

Protein structures in the context of time and space by mass spectrometry

Datasets

Sequential digestion with Trypsin and Elastase in cross-linking mass spectrometry

Cite this