Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers

Article abstract of DOI:10.1002/anie.201809149

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, transie

Full text of DOI:10.1002/anie.201809149

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Accepted Article
Title: Rapid Mapping of Protein Interactions Using Tag-Transfer
Photocrosslinkers
Authors: Jim Horne, Martin Walko, anton calabrese, mark levenstein,
david brockwell, nikil kapur, andrew wilson, and Sheena E.
Radford
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201809149
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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*, and Sheena. E. Radford*
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 methanethiosulphonate (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 initially 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).
complexity, poor fragmentation efficiency, and the increased
search space associated with the large numbers of peptide
combinations.[ Bulky crosslinking reagents may also perturb
native interactions or create new aberrant interactions.[9]
Furthermore, many biological processes, e.g. protein folding,
binding and conformational changes, occur on short timescales.
Consequently, crosslinks detected for a dynamic/non-equilibrium
system report the average of multiple states or are dominated by
the longest-lived state of all those populated. Photocrosslinkers
such as diazirines can address this challenge and have been
used previously to tag Cys residues in peptides.[ Diazirine-
generated carbenes react with proteins in ns,[11] yet long
irradiation times (minutes to hours[10b, 12]) are often required to
generate acceptable crosslink yields due to the use of low
intensity lamps.
2]
10]
Here we exploit a “tag and transfer” approach[13] to develop
two new XL-MS reagents and a workflow that enables crosslink
identification for both well-defined and dynamic protein-protein
interactions (PPIs). These heterobifunctional reagents comprise
a methanethiosulphonate (MTS) group for specific attachment
onto a single Cys residue introduced into the “bait” protein,
creating a cleavable disulfide bond within the linker arm, and a
diazirine that crosslinks to a “target” protein (Fig. 1a,b, Fig. S1-
S2). Commercially available MTS-benzophenone-biotin tags
have been used in photoinduced-crosslinking (PI-XL), but these
bulky tags (752 Da) may perturb PPI interfaces.[14] In the reagents
described here, reduction of the disulfide bond-containing linker
arm between the crosslinked proteins leaves a thiol tag on the
target (87 or 204 Da in size) (Fig. 1b, Fig. S3). The modified
residue can then be alkylated (e.g. with iodoacetamide [IAA]) and
localised using MS protocols for identifying post-translational
modifications (PTMs),[2] or enriched before MS. We also describe
the construction of a 365 nm UV LED (Fig. 1c, Fig. S4a,b) which
enables crosslinking reactions to be completed in just 10 seconds
with marginal heating. We exemplify this methodology by
mapping two PPIs, each with a different binding mode: a complex
Chemical crosslinking-mass spectrometry (XL-MS) is a
powerful component of the structural biology toolkit.[1] XL-MS
[
2]
methods have been enhanced by new data analysis software,
[3]
[4]
[6]
cleavable crosslinkers, strategies for crosslink enrichment,
footprinting reagents,[ and structural modelling approaches.
5]
However, several challenges remain. For example, many XL-MS
reagents have limited chemical reactivities (e.g. N-succinimide
ester-based reagents)[7] restricting the residues for which
information can be obtained. Secondly, the low-abundance of
crosslinked products often necessitates enrichment prior to MS.[
Finally, crosslinked peptide identification is difficult due to spectral
8]
[*]
Jim E. Horne[+], Dr Antonio N. Calabrese[+], Dr David J Brockwell,
Prof Sheena E Radford
School of Molecular and Cellular Biology, Faculty of Biological
Sciences, University of Leeds, Leeds, LS2 9JT (UK)
E-mail: s.e.radford@leeds.ac.uk
between BID80-102 and MCL-1 (K
D
= 50 ± 20 nM),[15] where the
tight binding affinity is mediated by several key residues[
15a, 16]
and
Dr Martin Walko[+], Mark A Levenstein, Prof Andrew J. Wilson
School of Chemistry, University of Leeds, Leeds, LS2 9JT (UK)
E-mail: a.j.wilson@leeds.ac.uk
the dynamic chaperone/substrate complex Skp/OmpA (K
D
=
2
2 ± 16 nM),[17] in which the binding interface involves many
rapidly interconverting interactions.[
17-18]
Jim E. Horne[+], Dr Martin Walko[+], Dr Antonio N. Calabrese[+], Dr
David J Brockwell, Prof Andrew J Wilson, Prof Sheena E Radford
Astbury Centre for Structural Molecular Biology, University of Leeds,
Leeds, LS2 9JT (UK)
Mark A Levenstein, Nikil Kapur
School of Mechanical Engineering, University of Leeds, Leeds, LS2
9JT (UK)
[+
]
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
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Figure 2. (a) Densitometry quantification of the appearance of
OmpA(W7C)[MTS-diazirine] crosslinked to Skp vs. irradiation time. Inset:
expanded time axis to show full time course using the Hg-Xe lamp. Lines are
exponential fits to the data. Relative intensity of crosslinked product (%) was
calculated as the intensity of the crosslinked product band on an SDS-PAGE
gel at time t divided by the intensity at tfinal, assuming the maximal yield is
achieved when the graph plateaus (see example SDS-PAGE of the XL reaction
(bottom) and Fig. S4c). (b) Sample heating at irradiation times required to reach
15, 55 and 100 % maximal crosslinked product for the Hg-Xe and UV LED lamps.
n.d. = not determined.
Figure 1. (a) Structures of MTS-diazirine and MTS-TFMD. (b) Crosslinking
workflow schematic: A Cys-containing bait protein is conjugated with the
reagent (here MTS-diazirine). After adding the target protein, the sample is
irradiated with 365 nm UV light, revealing a carbene that reacts with the target.
Reductant is added, leaving a sulfhydryl tag on the target at the interaction site.
(c) Image of the UV LED lamp and custom-built acrylic chip comprising a 33 µL
sample well. See also Fig. S4a,b.
MTS-diazirine and MTS-trifluoromethyl phenyl diazirine
MTS-TFMD) (Fig. 1a, Fig. S1) were chosen as photoactivatable
The human apoptotic regulatory pair BID/MCL-1 is a target
for cancer drug discovery.[22] The BH3 binding domain of BID
(
groups due to the superior performance of diazirines in
comparative PI-XL studies,[12c, 19] their small size, and rapid,
indiscriminate reactivity (Fig. S2).[9, 11] Both diazirine- and TFMD-
containing crosslinkers were synthesised as the photochemistry
of TFMD leads to higher crosslinking efficiency, but it is bulkier.[
In the bait, a unique solvent exposed Cys for crosslinker
conjugation is placed by mutagenesis or synthesis. In
proteins/peptides which lack Cys, or contain buried Cys, this is
straightforward. When a solvent exposed Cys is already present
in the bait this can be exploited, or substituted with Ala or Ser, and
a new Cys introduced in a location of interest. Knowing the
location of the Cys on one partner in the PPI reduces the MS/MS
(BID80-102) adopts a helix on binding to a surface groove on MCL-
[15b, 16]
1 (Fig. 3a).
Five single Cys variants of BID80-102 (Fig. 3a)
were synthesised (see Supporting Information), each with a
unique Cys residue in a different position. Subsequently, the
peptides were tagged with MTS-diazirine or MTS-TFMD. The
modified peptides bound MCL-1 with high affinity, although those
with the tagged residues in the centre of BID80-102 (I86C and V93C)
had a reduced EC50 (Fig. 3b, Table S1). Crosslinking of the BID80-
102 peptides with MCL-1 was achieved by UV LED irradiation.
Crosslinked complexes were detected by SDS-PAGE (Fig. 3c).
The absolute crosslinking efficiencies varied from 9-40 % for
MTS-diazirine, with reduced efficiency for lower affinity variants
(Table S1). Absolute crosslinking efficiencies were higher for the
MTS-TFMD tagged peptides (26-53 %) (Table S1), as expected
given the greater yield of TFMD-derived carbenes.[20] Gel bands
of the crosslinked complexes were excised and the linker arm
cleaved by reduction of the disulfide (Fig. 1b) releasing MCL-1
bearing the transferred thiol-containing tag at the interaction sites.
The free thiol in each tag was capped by alkylation with IAA and
the protein digested in-gel with trypsin (see Supporting
Information). Peptides modified with the transferred tag were
identified by LC-MS/MS. This allowed BID80-102 to be mapped into
the MCL-1 binding groove unambiguously, in agreement with the
known ‘bind-and-fold’ interaction[23] and NMR/X-ray structures of
the complex (Fig. 3d, Tables S2 and S3, Figs. S6-S8).[15b, 16] Both
crosslinkers gave similar results (Fig. S6), demonstrating that the
20]
2
search space from ntot to ntarget (where ntot is the number of
crosslinkable residues in the bait and target – for diazirines, this
is every residue in the proteins – and ntarget is the number of
residues in the target). Here, we introduce a unique Cys by
mutagenesis into the E. coli outer membrane protein OmpA, while
for the BID80-102 peptide, which comprises the binding domain of
the pro-apoptotic protein BID, Cys was introduced via solid-phase
peptide synthesis. Importantly, the reactivity of the photoactivated
diazirine places no restrictions on the amino acids in the target
protein that can be detected once a complex is formed.
To enhance diazirine photoactivation efficiency we
constructed a 365 nm LED lamp (Fig. 1c, Fig. S4a,b) with a
-2
nominal flux density of 15 W⋅cm . We designed devices to
irradiate samples in 0.2 mL to 1.5mL tubes, or in acrylic chips[
21]
with 33 μL sample wells (Fig. 1c, Fig. S5). Using this LED platform, bulkier TFMD-diazirine can be used even when sidechain
maximal XL yields were achieved in 10 seconds, with an
associated heating of < 2 °C (Fig. 2a,b, Fig. S4c). By contrast, a
interdigitation drives association. Crosslinked positions on MCL-1
were quantified by MS/MS from each Cys residue introduced into
BID80-102 labelled with MTS-diazirine or MTS-TFMD. This revealed
that crosslinking yields were greater when the Cα-Cα Euclidian
distance of the residues in the complex was <10 Å, but crosslinks
could be detected for distances up to 15 Å (Figs. S9-S10).
6
W Hg-Xe lamp achieved maximal yields after ca. 20 minutes
with heating of 10 °C (Fig. 2a,b, Fig. S4c), consistent with
previous reports.[10b, 12a, 12b] The UV LED design thus improves the
reaction rate 130-fold, and decreases heating 6-fold.
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Figure 3. (a) Sequence of WT BID80-102. Five Cys variants
of BID80-102 were labelled with MTS-diazirine or MTS-
TFMD. Cys-substituted amino acids are shown as
coloured spheres in the peptide structure and in the same
colour in the sequence above. (b) Inhibitory potency (EC50
)
of BID80-102 labelled with MTS-diazirine (left) or MTS-TFMD
(
(
right) to MCL-1 measured by fluorescence anisotropy
EC50 values are shown in Table S1). (c) SDS-PAGE of
BID80-102 labelled with MTS-diazirine (top) or MTS-TFMD
(
(
bottom) crosslinked to MCL-1. (d) Residues of MCL-1
magenta on a grey ribbon) that crosslinked to at least one
BID80-102 peptide labelled with MTS-diazirine (left) or MTS-
TFMD (right). BID80-102 is shown in cyan and residues
substituted as Cys are coloured as in (a) (PDB ID:
KBW[ ). See also Figs. S6-S8, Tables S2, S3.
16]
2
We next tested the ability of our workflow and tag transfer
reagents to study a protein complex stabilised by transient
interactions. Chaperone-client binding often involves a dynamic
interaction of chaperones with an unfolded/partially folded client
protein to aid folding or prevent aggregation.[24] These interfaces
are challenging to map using crosslinking since the interactions
can be dynamic and diffuse. Here, we used the interaction
between the periplasmic chaperone Skp from E. coli, and a β-
barrel outer membrane protein substrate, OmpA, as a model for
this type of PPI.[25] Skp, a homotrimer, has a jellyfish-like structure
resulting in a cage in which substrates are sequestered (Fig.
[
25b, 26]
[17]
4a).
Skp binds its OMP substrates with nM affinity, similar
to that of the well-defined BID80-102:MCL-1 complex.[15] However,
Skp-bound OmpA is in a ‘fluid-globule’ state that forms many
weak and transient interactions in the Skp cage.[17-18]
Two single Cys variants of OmpA were generated, W7C and
T144C, located in the β-barrel and a surface exposed loop in
folded OmpA, respectively.[
18]
MTS-diazirine or MTS-TFMD
conjugation of both variants was efficient (Fig. S11), and did not
prevent folding (Fig. S12). The Skp/OmpA complex was
assembled by dilution of urea-denatured OmpA into a Skp-
containing solution, and PI-XL was then performed. The
crosslinked Skp/OmpA complex was detected by non-reducing
SDS-PAGE as a single band with an apparent mass of ∼70 kDa
Figure 4. (a) Model of the Skp/OmpA complex. Trimeric Skp (PDB ID: 1U2M[26]
)
is shown in cyan, pink and green. Collapsed OmpA (yellow) is shown in the Skp
cavity.[
25b]
(b) Non-reducing SDS-PAGE to show enrichment of crosslinked
(
Fig. 4b). Four non-overlapping positions from OmpA(T144C) to
Skp/OmpA. The crosslinked Skp/OmpA(T144C)[MTS-diazirine] complex (lane
1, XL) was cleaved with DTT. The constituent proteins contain free thiols so can
be bound to thiopropyl Sepharose 6B. Unbound material was removed by
washing and the captured material eluted by adding DTT. (c) Structure of Skp
residues within Skp’s ‘cage’ (Fig. 4d (top); Fig. S13) could be
identified by in-gel digestion of the crosslinked proteins followed
by LC-MS/MS (Fig. 5, Method 1). This result was surprising since
it is inconsistent with previous studies which have shown that
OmpA tumbles dynamically on a sub-ms timescale within Skp.[
We reasoned that the lack of modified sites resulted from the
relatively low abundance of these peptides in the Skp-OmpA
complex, rather than reflecting a specific interaction surface
involving these four sites. Given that each Skp trimer binds a
single OmpA, and each OmpA contains only a single crosslinker,
then only one modified peptide would result from each 70 kDa
Skp:OmpA complex.
with crosslinked residues from
a pooled dataset of OmpA(T144C)[MTS-
diazirine] (Fig. S13, Table S4). One monomer in the Skp trimer is highlighted.
In c and d, red represents residue-level information and blue represents
crosslinks localised within a 2 or more residue region. (d) Crosslinked residues
identified by the enrichment methods shown in Fig. 5 for OmpA(T144C)[MTS-
diazirine]. The lower plot shows the total number of unique sites identified by
the three enrichment strategies shown in Fig. 5. Representative mass spectra
of modified Skp peptides are shown in Figs. S15-S16 and a list of crosslinked
peptides is in Tables S4-S5.
18]
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To test this conclusion we developed a protocol to enrich
chemically modified Skp, removing background peptides arising
from OmpA and unmodified Skp (Fig. 5). We used thiopropyl
Sepharose 6B beads to purify Skp monomers containing the thiol
fragment obtained from the tag-transfer reaction and the Cys-
containing OmpA bait. The thiol-containing proteins were then
eluted from the resin, separated by SDS-PAGE and trypsinised
in-gel (Method 2). Alternatively, trypsin digestion was performed
on-bead[27] and unbound peptides removed by washing the resin
prior to elution (Method 3). Using Method 2 the number of
modified peptides detected increased >2-fold (from 6 to 14), whilst
Method 3 yielded a further 2.5-fold increase in the number of
modified peptides identified (from 14 to 35) (Fig. 4d, Fig. S13), a
~6-fold increase in detection over Method 1. No significant
sidechain bias was observed for either crosslinker (Fig. S14),
[
9,
consistent with the reactivity of the diazirine-derived carbenes.
1
9b]
Similar crosslinking sites were observed for OmpA(W7C) and
OmpA(T144C) using both crosslinkers (Figs. S13-S14). Using
these enrichment protocols modifications on Skp were identified
all around the internal cavity (Fig. 4c), consistent with OmpA
tumbling randomly in Skp.[18] The ability to enrich crosslinked
peptides using our tag-transfer protocol, combined with the
promiscuous reactivity of diazirines, exemplifies the power of the
technique to monitor even the most dynamic of protein interfaces
(
Fig. 4c & d).
In summary, we have demonstrated that Cys-containing
variants of a bait protein conjugated with MTS-diazirine or MTS-
TFMD-based tag-transfer crosslinkers can map PPIs in both well-
defined and dynamic interfaces. Since the location of the crosslink
in the bait protein is known and only the transferred tag is
considered in downstream analysis, the background from bait
peptides is removed and the search space for crosslinked
products is reduced. These features are particularly important as
target protein size increases. The workflow described is simple to
implement, only requiring the appropriate crosslinking reagents,
LC-MS/MS and proteomics software for mapping PTMs.
Enrichment and digestion of modified peptides enables low
protein concentrations to be used, minimising the possibility of
aggregation or other aberrant interactions. Additionally, the
custom UV LED platform enables PI-XL on a 10 second timescale,
not possible with arc-based lamps, and only previously achieved
using pulsed lasers in solution or in the gas phase.[5, 10a] The low
cost (~ $300) and simplicity of our UV LED system makes these
timescales accessible to any researcher. Our workflow thus
opens the door to time-resolved XL on the second timescale
without the need for expensive lasers and enables the study of
conformational changes within dynamic protein complexes versus
time.
Figure 5. Enrichment and analysis strategies for MTS-diazirine and MTS-TFMD.
Irradiation at 365 nm results in crosslinking of bait and target proteins (n.b. low
reaction efficiencies mean that uncrosslinked material will remain). Enrichment
can be performed using one of three methods (see the text). MS analysis of the
peptides is then performed, and the data searched to identify the
peptides/residues modified with the crosslinking reagent (with the free thiol
capped by reaction with IAA).
Acknowledgements
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/M01151/1,
BB/K000659/1, BB/N007603/1 and BB/M012573/1), EPSRC
(
(
EP/N035267/1 and EP/N013573/1) and Wellcome Trust
208385/Z/17/Z) are acknowledged for their support. MAL is
supported by a Leeds International Research Scholarship. NK
acknowledges the support of RAEng and GSK. SER
acknowledges funding from the European Research Council
under the European Union’s Seventh Framework Programme
grant FP7.2007-2013/grant agreement number 322408.
2
010, 17, 1325-1333; fA. Sinz, Angew Chem Int Ed 2018, 57,
Keywords: chemical crosslinking • mass spectrometry • diazo
6390-6396.
compounds • photoaffinity labeling • protein-protein interactions
[2]
S. Yilmaz, G. A. Shiferaw, J. Rayo, A. Economou, L. Martens, E.
Vandermarliere, Mass Spectrom Rev 2018, 37, 738-749.
[
3]
aC. Iacobucci, C. Piotrowski, A. Rehkamp, C. H. Ihling, A. Sinz, J
Am Soc Mass Spectrom 2018; bM. Q. Muller, F. Dreiocker, C. H.
Ihling, M. Schafer, A. Sinz, Anal Chem 2010, 82, 6958-6968; cY.
Yang, H. Song, D. He, S. Zhang, S. Dai, X. Xie, S. Lin, Z. Hao, H.
Zheng, P. R. Chen, Nat Protoc 2017, 12, 2147-2168; dC.
Iacobucci, M. Gotze, C. Piotrowski, C. Arlt, A. Rehkamp, C. Ihling,
C. Hage, A. Sinz, Anal Chem 2018, 90, 2805-2809.
[1]
aM. Schneider, A. Belsom, J. Rappsilber, Trends Biochem Sci
2
2
018, 43, 157-169; bA. N. Calabrese, S. E. Radford, Methods
018, 147, 187-205; cJ. Gubbens, E. Ruijter, L. E. V. de Fays, J.
M. A. Damen, B. de Kruijff, M. Slijper, D. T. S. Rijkers, R. M. J.
Liskamp, A. I. P. M. de Kroon, Chem Biol 2009, 16, 3-14; dO.
Pavlova, J. H. Peterson, R. Ieva, H. D. Bernstein, Proc Natl Acad
Sci U S A 2013, 110, E938-947; eC. R. Braun, J. Mintseris, E.
Gavathiotis, G. H. Bird, S. P. Gygi, L. D. Walensky, Chem Biol
[4]
D. Tan, Q. Li, M. J. Zhang, C. Liu, C. Ma, P. Zhang, Y. H. Ding, S.
B. Fan, L. Tao, B. Yang, X. Li, S. Ma, J. Liu, B. Feng, X. Liu, H. W.
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201809149
COMMUNICATION
Wang, S. M. He, N. Gao, K. Ye, M. Q. Dong, X. Lei, eLife 2016, 5,
e12509.
aL. Manzi, A. S. Barrow, D. Scott, R. Layfield, T. G. Wright, J. E.
Moses, N. J. Oldham, Nat Commun 2016, 7, 13288; bL. Manzi, A.
S. Barrow, J. T. S. Hopper, R. Kaminska, C. Kleanthous, C. V.
Robinson, J. E. Moses, N. J. Oldham, Angew Chem Int Ed 2017,
[14]
[15]
R. Ahrends, J. Kosinski, D. Kirsch, L. Manelyte, L. Giron-Monzon,
L. Hummerich, O. Schulz, B. Spengler, P. Friedhoff, Nucleic Acids
Res 2006, 34, 3169-3180.
aE. F. Lee, G. Dewson, M. Evangelista, A. Pettikiriarachchi, G. J.
Gold, H. R. Zhu, P. M. Colman, W. D. Fairlie, J Biol Chem 2014,
289, 36001-36017; bJ. A. Miles, D. J. Yeo, P. Rowell, S.
Rodriguez-Marin, C. M. Pask, S. L. Warriner, T. A. Edwards, A. J.
Wilson, Chem Sci 2016, 7, 3694-3702.
Q. Liu, T. Moldoveanu, T. Sprules, E. Matta-Camacho, N. Mansur-
Azzam, K. Gehring, J Biol Chem 2010, 285, 19615-19624.
J. Qu, C. Mayer, S. Behrens, O. Holst, J. H. Kleinschmidt, J Mol
Biol 2007, 374, 91-105.
[5]
56, 14873-14877.
[6]
aA. Politis, C. Schmidt, J Proteomics 2018, 175, 34-41; bM.
Ferber, J. Kosinski, A. Ori, U. J. Rashid, M. Moreno-Morcillo, B.
Simon, G. Bouvier, P. R. Batista, C. W. Muller, M. Beck, M. Nilges,
Nat Methods 2016, 13, 515-520.
aS. Kalkhof, A. Sinz, Anal Bioanal Chem 2008, 392, 305-312; bS.
Madler, C. Bich, D. Touboul, R. Zenobi, J Mass Spectrom 2009,
[16]
[17]
[18]
[19]
[
7]
8]
B. M. Burmann, C. Wang, S. Hiller, Nat Struct Mol Biol 2013, 20,
1265-1272.
44, 694-706.
[
aR. Fritzsche, C. H. Ihling, M. Gotze, A. Sinz, Rapid Commun
Mass Spectrom 2012, 26, 653-658; bR. M. Kaake, X. Wang, A.
Burke, C. Yu, W. Kandur, Y. Yang, E. J. Novtisky, T. Second, J.
Duan, A. Kao, S. Guan, D. Vellucci, S. D. Rychnovsky, L. Huang,
Mol Cell Proteomics 2014, 13, 3533-3543; cA. Leitner, R. Reischl,
T. Walzthoeni, F. Herzog, S. Bohn, F. Forster, R. Aebersold, Mol
Cell Proteomics 2012, 11, M111.014126; dX. Tang, J. E. Bruce,
Mol Biosyst 2010, 6, 939-947.
G. W. Preston, A. J. Wilson, Chem Soc Rev 2013, 42, 3289-3301.
aY. Liu, Z. Ramey, F. Turecek, Chem. Eur. J 2018, 24, 9259-9263;
bJ. Liang, L. Zhang, X. L. Tan, Y. K. Qi, S. Feng, H. Deng, Y. Yan,
J. S. Zheng, L. Liu, C. L. Tian, Angew Chem Int Ed 2017, 56,
aL. Dubinsky, B. P. Krom, M. M. Meijler, Bioorg Med Chem 2012,
20, 554-570; bM. Suchanek, A. Radzikowska, C. Thiele, Nat
Methods 2005, 2, 261-267.
J. Brunner, H. Senn, F. M. Richards, J Biol Chem 1980, 255, 3313-
3318.
A. Liga, J. A. S. Morton, M. Kersaudy-Kerhoas, Microfluid
Nanofluid 2016, 20, 164.
G. Lessene, P. E. Czabotar, P. M. Colman, Nat Rev Drug Discov
2008, 7, 989-1000.
[20]
[21]
[22]
[23]
[9]
[10]
J. M. Rogers, V. Oleinikovas, S. L. Shammas, C. T. Wong, D. De
Sancho, C. M. Baker, J. Clarke, Proc Natl Acad Sci U S A 2014,
111, 15420-15425.
2
744-2748.
[24]
[25]
D. Balchin, M. Hayer-Hartl, F. U. Hartl, Science 2016, 353,
aac4354.
[
11]
12]
A. Admasu, A. D. Gudmundsdottir, M. S. Platz, D. S. Watt, S.
Kwiatkowski, P. J. Crocker, J. Chem. Soc., Perkin Trans. 2 1998,
aB. Schiffrin, D. J. Brockwell, S. E. Radford, BMC Biol 2017, 15,
123; bB. Schiffrin, A. N. Calabrese, P. W. A. Devine, S. A. Harris,
A. E. Ashcroft, D. J. Brockwell, S. E. Radford, Nat Struct Mol Biol
2016, 23, 786-793.
T. A. Walton, M. C. Sousa, Mol Cell 2004, 15, 367-374.
J. Guo, M. J. Gaffrey, D. Su, T. Liu, D. G. Camp, 2nd, R. D. Smith,
W. J. Qian, Nat Protoc 2014, 9, 64-75.
1093-1099.
[
aT. Yang, Z. Liu, X. D. Li, Chem Sci 2015, 6, 1011-1017; bM.
Zhang, S. Lin, X. Song, J. Liu, Y. Fu, X. Ge, X. Fu, Z. Chang, P. R.
Chen, Nat Chem Biol 2011, 7, 671-677; cG. W. Preston, S. E.
Radford, A. E. Ashcroft, A. J. Wilson, ACS Chem Biol 2014, 9,
[26]
[27]
761-768.
[13]
E. Nakata, Y. Koshi, E. Koga, Y. Katayama, I. Hamachi, J Am
Chem Soc 2005, 127, 13253-13261.
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Angewandte Chemie International Edition
10.1002/anie.201809149
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Jim E. Horne, Martin Walko, Antonio N.
Calabrese, Mark A. Levenstein, David J.
Brockwell, Nikil Kapur, Andrew J.
Wilson*, Sheena. E. Radford*
Page No. – Page No.
Fast, enrichable photocrosslinking: Photocrosslinking reagents with
methanethiosulphonate and diazirine functionalities have been developed. After
crosslinking and reduction, a thiol tag stays at the interaction site, to be located by
mass spectrometry and used for enrichment of even low abundance crosslinks. A
UV LED irradiation platform reduces crosslinking times to 10 seconds. This
approach improves the temporal resolution of crosslinking and enables facile
mapping of protein interfaces.
Rapid Mapping of Protein Interactions
Using Tag-Transfer
Photocrosslinkers
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