Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers

Jim E. Horne; Martin Walko; Antonio N. Calabrese; Mark A. Levenstein; David J. Brockwell; Nikil Kapur; Andrew J. Wilson; Sheena E. Radford

doi:10.1002/anie.201809149

Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers

Jim E. Horne, Martin Walko, Antonio N. Calabrese, Mark A. Levenstein, David J. Brockwell, Nikil Kapur, Andrew J. Wilson^*, Sheena E. Radford

^*Corresponding author for this work

Chemistry

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

38 Citations (Scopus)

Abstract

Analysing protein complexes by chemical crosslinking-mass spectrometry (XL-MS) is limited by the side-chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue-unbiased diazirine-based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol-containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this “tag and transfer” approach is demonstrated using a well-defined peptide/protein regulatory interaction (BID_80-102/MCL-1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA).

Original language	English
Pages (from-to)	16688-16692
Number of pages	5
Journal	Angewandte Chemie - International Edition
Volume	57
Issue number	51
DOIs	https://doi.org/10.1002/anie.201809149
Publication status	Published - 17 Dec 2018

Bibliographical note

Funding Information:
We thank Nasir Khan and Rachel George for technical assistance; Dr Thomas Edwards and Pallavi Ramsahye for MCL-1; Dr Bob Schiffrin for the collapsed model of tOmpA; and Patrick Mason for use of an IR camera. His-tagged Skp plasmid was kindly provided by S. Hiller (University of Basel, Switzerland). Full-length OmpA plasmid was kindly provided by K. Fleming (John Hopkins University, USA). The BBSRC (BB/M011151/1, BB/K000659/1, BB/N007603/1 and BB/ M012573/1, BB/P000037/1), EPSRC (EP/N035267/1 and EP/ N013573/1) and Wellcome Trust (208385/Z/17/Z) are acknowledged for their support. M.A.L. is supported by a Leeds International Research Scholarship. N.K. acknowledges the support of RAEng and GSK. S.E.R. acknowledges funding from the European Research Council under the European Union≫s Seventh Framework Programme grant FP7.2007-2013/grant agreement number 322408.

Funding Information:
We thank Nasir Khan and Rachel George for technical assistance; Dr Thomas Edwards and Pallavi Ramsahye for MCL-1; Dr Bob Schiffrin for the collapsed model of tOmpA; and Patrick Mason for use of an IR camera. His-tagged Skp plasmid was kindly provided by S. Hiller (University of Basel, Switzerland). Full-length OmpA plasmid was kindly provided by K. Fleming (John Hopkins University, USA). The BBSRC (BB/M011151/1, BB/K000659/1, BB/N007603/1 and BB/M012573/1, BB/P000037/1), EPSRC (EP/N035267/1 and EP/N013573/1) and Wellcome Trust (208385/Z/17/Z) are acknowledged for their support. M.A.L. is supported by a Leeds International Research Scholarship. N.K. acknowledges the support of RAEng and GSK. S.E.R. acknowledges funding from the European Research Council under the European Union's Seventh Framework Programme grant FP7.2007-2013/grant agreement number 322408.

Publisher Copyright:
© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Keywords

chemical crosslinking
diazo compounds
mass spectrometry
photoaffinity labeling
protein–protein interactions

ASJC Scopus subject areas

Catalysis
General Chemistry

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10.1002/anie.201809149

Cite this

@article{5e6b67160802419c8d98d742cd336339,

title = "Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers",

abstract = "Analysing protein complexes by chemical crosslinking-mass spectrometry (XL-MS) is limited by the side-chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue-unbiased diazirine-based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol-containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this “tag and transfer” approach is demonstrated using a well-defined peptide/protein regulatory interaction (BID80-102/MCL-1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA).",

keywords = "chemical crosslinking, diazo compounds, mass spectrometry, photoaffinity labeling, protein–protein interactions",

author = "Horne, {Jim E.} and Martin Walko and Calabrese, {Antonio N.} and Levenstein, {Mark A.} and Brockwell, {David J.} and Nikil Kapur and Wilson, {Andrew J.} and Radford, {Sheena E.}",

note = "Funding Information: We thank Nasir Khan and Rachel George for technical assistance; Dr Thomas Edwards and Pallavi Ramsahye for MCL-1; Dr Bob Schiffrin for the collapsed model of tOmpA; and Patrick Mason for use of an IR camera. His-tagged Skp plasmid was kindly provided by S. Hiller (University of Basel, Switzerland). Full-length OmpA plasmid was kindly provided by K. Fleming (John Hopkins University, USA). The BBSRC (BB/M011151/1, BB/K000659/1, BB/N007603/1 and BB/ M012573/1, BB/P000037/1), EPSRC (EP/N035267/1 and EP/ N013573/1) and Wellcome Trust (208385/Z/17/Z) are acknowledged for their support. M.A.L. is supported by a Leeds International Research Scholarship. N.K. acknowledges the support of RAEng and GSK. S.E.R. acknowledges funding from the European Research Council under the European Union≫s Seventh Framework Programme grant FP7.2007-2013/grant agreement number 322408. Funding Information: We thank Nasir Khan and Rachel George for technical assistance; Dr Thomas Edwards and Pallavi Ramsahye for MCL-1; Dr Bob Schiffrin for the collapsed model of tOmpA; and Patrick Mason for use of an IR camera. His-tagged Skp plasmid was kindly provided by S. Hiller (University of Basel, Switzerland). Full-length OmpA plasmid was kindly provided by K. Fleming (John Hopkins University, USA). The BBSRC (BB/M011151/1, BB/K000659/1, BB/N007603/1 and BB/M012573/1, BB/P000037/1), EPSRC (EP/N035267/1 and EP/N013573/1) and Wellcome Trust (208385/Z/17/Z) are acknowledged for their support. M.A.L. is supported by a Leeds International Research Scholarship. N.K. acknowledges the support of RAEng and GSK. S.E.R. acknowledges funding from the European Research Council under the European Union's Seventh Framework Programme grant FP7.2007-2013/grant agreement number 322408. Publisher Copyright: {\textcopyright} 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.",

year = "2018",

month = dec,

day = "17",

doi = "10.1002/anie.201809149",

language = "English",

volume = "57",

pages = "16688--16692",

journal = "Angewandte Chemie - International Edition",

issn = "1433-7851",

publisher = "Wiley-VCH Verlag",

number = "51",

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T1 - Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers

AU - Horne, Jim E.

AU - Walko, Martin

AU - Calabrese, Antonio N.

AU - Levenstein, Mark A.

AU - Brockwell, David J.

AU - Kapur, Nikil

AU - Wilson, Andrew J.

AU - Radford, Sheena E.

N1 - Funding Information: We thank Nasir Khan and Rachel George for technical assistance; Dr Thomas Edwards and Pallavi Ramsahye for MCL-1; Dr Bob Schiffrin for the collapsed model of tOmpA; and Patrick Mason for use of an IR camera. His-tagged Skp plasmid was kindly provided by S. Hiller (University of Basel, Switzerland). Full-length OmpA plasmid was kindly provided by K. Fleming (John Hopkins University, USA). The BBSRC (BB/M011151/1, BB/K000659/1, BB/N007603/1 and BB/ M012573/1, BB/P000037/1), EPSRC (EP/N035267/1 and EP/ N013573/1) and Wellcome Trust (208385/Z/17/Z) are acknowledged for their support. M.A.L. is supported by a Leeds International Research Scholarship. N.K. acknowledges the support of RAEng and GSK. S.E.R. acknowledges funding from the European Research Council under the European Union≫s Seventh Framework Programme grant FP7.2007-2013/grant agreement number 322408. Funding Information: We thank Nasir Khan and Rachel George for technical assistance; Dr Thomas Edwards and Pallavi Ramsahye for MCL-1; Dr Bob Schiffrin for the collapsed model of tOmpA; and Patrick Mason for use of an IR camera. His-tagged Skp plasmid was kindly provided by S. Hiller (University of Basel, Switzerland). Full-length OmpA plasmid was kindly provided by K. Fleming (John Hopkins University, USA). The BBSRC (BB/M011151/1, BB/K000659/1, BB/N007603/1 and BB/M012573/1, BB/P000037/1), EPSRC (EP/N035267/1 and EP/N013573/1) and Wellcome Trust (208385/Z/17/Z) are acknowledged for their support. M.A.L. is supported by a Leeds International Research Scholarship. N.K. acknowledges the support of RAEng and GSK. S.E.R. acknowledges funding from the European Research Council under the European Union's Seventh Framework Programme grant FP7.2007-2013/grant agreement number 322408. Publisher Copyright: © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

PY - 2018/12/17

Y1 - 2018/12/17

N2 - Analysing protein complexes by chemical crosslinking-mass spectrometry (XL-MS) is limited by the side-chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue-unbiased diazirine-based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol-containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this “tag and transfer” approach is demonstrated using a well-defined peptide/protein regulatory interaction (BID80-102/MCL-1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA).

AB - Analysing protein complexes by chemical crosslinking-mass spectrometry (XL-MS) is limited by the side-chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue-unbiased diazirine-based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol-containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this “tag and transfer” approach is demonstrated using a well-defined peptide/protein regulatory interaction (BID80-102/MCL-1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA).

KW - chemical crosslinking

KW - diazo compounds

KW - mass spectrometry

KW - photoaffinity labeling

KW - protein–protein interactions

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U2 - 10.1002/anie.201809149

DO - 10.1002/anie.201809149

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ER -

Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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