Synthesis of two new enrichable and MS-cleavable cross-linkers to define protein-protein interactions by mass spectrometry

Article abstract of DOI:10.1039/c5ob00488h

The cross-linking Mass Spectrometry (XL-MS) technique extracts structural information from protein complexes without requiring highly purified samples, crystallinity, or large amounts of material. However, there are challenges to applying the technique to protein complexes in vitro, and those challenges become more daunting with in vivo experiments. Issues include effective detection and identification of cross-linked peptides from complex mixtures. While MS-cleavable cross-linkers facilitate the sequencing and identification of cross-linked peptides, enrichable cross-linkers increase their detectability by allowing their separation from non-cross-linked peptides prior to MS analysis. Although a number of cross-linkers with single functionality have been developed in recent years, an ideal reagent would incorporate both capabilities for XL-MS studies. Therefore, two new cross-linkers have been designed and prepared that incorporate an azide (azide-A-DSBSO) or alkyne (alkyne-A-DSBSO) to enable affinity purification strategies based on click chemistry. The integration of an acid cleavage site next to the enrichment handle allows easy recovery of cross-linked products during affinity purification. In addition, these sulfoxide containing cross-linking reagents possess robust MS-cleavable bonds to facilitate fast and easy identification of cross-linked peptides using MS analysis. Optimized, gram-scale syntheses of these cross-linkers have been developed and the azide-A-DSBSO cross-linker has been evaluated with peptides and proteins to demonstrate its utility in XL-MS analysis.

Full text of DOI:10.1039/c5ob00488h

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Synthesis of two new enrichable and MS-cleavable
cross-linkers to define protein–protein interactions
by mass spectrometry†
Cite this: DOI: 10.1039/c5ob00488h
Anthony M. Burke,a Wynne Kandur,a Eric J. Novitsky,a Robyn M. Kaake,b Clinton Yu,b
Athit Kao,b Danielle Vellucci,a Lan Huangb and Scott D. Rychnovsky*a
The cross-linking Mass Spectrometry (XL-MS) technique extracts structural information from protein
complexes without requiring highly purified samples, crystallinity, or large amounts of material. However,
there are challenges to applying the technique to protein complexes in vitro, and those challenges
become more daunting with in vivo experiments. Issues include effective detection and identification of
cross-linked peptides from complex mixtures. While MS-cleavable cross-linkers facilitate the sequencing
and identification of cross-linked peptides, enrichable cross-linkers increase their detectability by allowing
their separation from non-cross-linked peptides prior to MS analysis. Although a number of cross-linkers
with single functionality have been developed in recent years, an ideal reagent would incorporate both
capabilities for XL-MS studies. Therefore, two new cross-linkers have been designed and prepared that
incorporate an azide (azide-A-DSBSO) or alkyne (alkyne-A-DSBSO) to enable affinity purification strat-
egies based on click chemistry. The integration of an acid cleavage site next to the enrichment handle
allows easy recovery of cross-linked products during affinity purification. In addition, these sulfoxide con-
taining cross-linking reagents possess robust MS-cleavable bonds to facilitate fast and easy identification
of cross-linked peptides using MS analysis. Optimized, gram-scale syntheses of these cross-linkers have
been developed and the azide-A-DSBSO cross-linker has been evaluated with peptides and proteins to
demonstrate its utility in XL-MS analysis.
Received 11th March 2015,
Accepted 20th March 2015
DOI: 10.1039/c5ob00488h
www.rsc.org/obc
Most proteins act in association with other proteins to form amino acid resolution.4 The advantages of cross-linking
protein complexes stably or transiently in cells, and mapping studies include small sample size, robust tolerance for size
these interactions is essential to understand their cellular and environment of the protein complex, instrument accessi-
functions. Protein complexes represent functional entities that bility, and the speed of handling and data collection. Although
are often difficult to analyze using conventional structural successful, inherent limitations in current XL-MS strategies
tools due to their heterogeneous and dynamic nature. require further developments to enable MS detection and
Recently, cross-linking Mass Spectrometry (XL-MS) has been identification of cross-linked peptides with better efficiency,
recognized as a valuable tool for the structural analysis of accuracy, sensitivity and speed. Among various approaches
protein assemblies,1 which can be used alone and in combi- to improve existing XL-MS workflow,5 developing new cross-
nation with other techniques.1,2 In addition to in vitro studies, linking reagents holds the greatest promise towards the
XL-MS approaches have been extended to capture protein ultimate goal of mapping protein–protein interactions in
interactions in living cells.3 Identification of cross-linked pep- living cells at the systems level. We report the chemical syn-
tides by MS analysis can provide distance constraints to assist thesis of two new cross-linking agents whose effectiveness has
computational modeling and yield structural information at recently been demonstrated for in vivo protein–protein
analysis.6
Unambiguous identification of cross-linked peptides can be
aDepartment of Chemistry, University of California, Irvine, CA 92697, USA.
E-mail: srychnov@uci.edu
greatly facilitated by the introduction of a MS cleavable bond
in a cross-linking reagent, which can fragment during collision
induced dissociation (CID) prior to peptide backbone break-
age.7 Previously, we have successfully developed a new class of
robust MS-cleavable reagents that contain labile C–S sulfoxide
bonds (e.g. DSSO (disuccinimidylsulfoxide), Fig. 1), and thus
bDepartments of Physiology & Biophysics, University of California, Irvine, CA 92697,
USA
†Electronic supplementary information (ESI) available: General synthetic
methods, experimental data, and spectral data are presented in pdf format. See
DOI: 10.1039/c5ob00488h
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with large, complex protein assemblies and especially when
studying protein–protein interactions in living cells. A strategy
to improve the sensitivity and efficiency of XL-MS analysis is to
incorporate an affinity purification handle into the cross-linker
itself. To this end, we have developed an azide-tagged cross-
linking reagent that allows the incorporation of an affinity
purification handle based on click chemistry for enriching
cross-linked peptides prior to MS analysis, thus improving
their detection and identification.18 In comparison to other
enrichment handles incorporated in cross-linking reagents,10
the azide group is advantageous as it is small, bioortho-
gonal, and click chemistry has been proven effective in enrich-
ing biological samples for various proteomic analyses
including cross-linking studies.11 In order to combine these
unique features in a multifunctional cross-linking reagent
that can advance current XL-MS workflow for studying
protein–protein interactions in vivo as well as in vitro, we have
developed a new class of low molecular weight, membrane
permeable, enrichable and MS-cleavable cross-linkers.
In order to explore the flexibility of using azide–alkyne click
chemistry in the XL-MS workflow, we have designed and syn-
thesized two new cross-linkers, i.e. azide-tagged and an alkyne-
tagged reagents as presented in Fig. 2. The structure of azide-
A-DSBSO (azide-tagged, acid-cleavable disuccinimidyl-bissulf-
oxide) 3 incorporates a number of important design elements.
The N-hydroxysuccinimidyl (NHS) esters are designed to react
with lysine side chains thus cross-linking the proteins in the
complex. The sulfoxide groups provide MS-cleavable bonds,
and because only one side of each sulfoxide has β-hydrogen
Fig. 1 CID cleavable cross-linker DSSO is based on the sulfoxide func-
tional group. The red arrow points to the bond that is broken during the
CID process. The workflow for cross-linking proteins is shown. After
protein cross-linking, trypsin digest generates the cross-linked peptide
for LC/MSn analysis. CID leads to selective cleavage of the bonds adja-
cent to the sulfoxide functional group.
enables fast and accurate identification of cross-linked
peptides using liquid chromatography-multistage tandem
mass spectrometry analysis (LC/MSn).8,9 With DSSO as an
example, this new XL-MS workflow involves protein DSSO
cross-linking, trypsin digestion of cross-linked proteins, and
LC/MSn analysis of resulting peptide mixtures. During MSn
analysis, the cross-linked peptides are first detected in MS1
and selected for subsequent MS2 analysis. The CID-fragmenta-
tion site, i.e. one of the C–S sulfoxide bonds, is selectively frag-
mented in MS2, allowing the physical separation of the two
DSSO cross-linked peptide constituents for subsequent
sequencing. The resulting peptide fragments in MS2 are then
analyzed in MS3 for unambiguous identification. The inte-
gration of these three types of MS data (MS1, MS2, MS3)
enables simplified analysis of DSSO cross-linked peptides with
improved speed and accuracy. This strategy has been demon-
strated to be effective in the structural analysis of purified
protein complexes in vitro.4
Fig. 2 Protein cross-linkers designed with CID cleavable sulfoxide
groups, azide (3) or alkyne (4) groups for click chemistry enrichment
strategies, and an acid labile acetal to facilitate affinity purification.
The analytical problem with effectively detecting and identi-
fying cross-linked peptides becomes much more daunting
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atoms, the elimination must take place regioselectively at the theses of these reagents are described in full along with foun-
outer C–S bond. The design incorporates an azide functional dational studies on the cross-linking effectiveness and LC/MSn
group to be used in click reactions with strained alkynes or in sequencing.
a copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC).12
The click and CuAAC reactions enable several strategies for
affinity purification, including direct coupling with alkyne or
Results and discussion
Synthesis of the cross-linkers 3 and 4
azide-functionalized beads or by linking with common affinity
ligands such as biotin.11 Finally, the azide portion of the mole-
cule is joined to the cross-linker with an acid labile acetal
bond, which can be cleaved under aqueous acidic conditions
to facilitate selective elution from an affinity column. The
same elements are incorporated into the alkyne-A-DSBSO
(alkyne-tagged, acid-cleavable disuccinimidyl-bissulfoxide) 4,
except that the azide functional group has been exchanged for
the complementary alkyne. One other design feature is that
both of these cross-linkers, prior to the introduction of the
sulfoxides, are achiral and exist as single stereoisomers. This
feature offers considerable simplification in the preparation
and analysis of the synthetic intermediates, and decreases the
chance of any stereoselective behavior in the crosslinking
environment. These reagents have been under investigation
for several years in our program and their applications in
mapping protein–protein interactions at the systems level in
living cells were recently described.6 In this report, the syn-
The original synthesis of azide-A-DSBSO 3 began with penta-
erythritol (5) and is presented in Scheme 1. Selective protection
of 5 as cyclohexanone acetal 6 represented an improvement of
the literature procedure.13 Mesylation followed by displace-
ment with potassium thioacetate produced the bis-thioacetate
8 in good overall yield. All three intermediates, 6–8, were crys-
talline solids that were isolated and purified on multi-gram
scale without chromatography. Thioacetate methanolysis and
Michael addition of 8 into methyl acrylate was accomplished
in a single step in the presence of triethylamine and methanol.
The key intermediate 9 was isolated in nearly quantitative yield
and used without further purification. Careful hydrolysis of
the cyclohexanone acetal using In(OTf)3 catalysis produced
86% of the diol 10 and 8% recovered starting material, which
were separated by chromatography. Incomplete hydrolysis of
the acetal was a recurring problem in this step when using
Scheme 1 Synthesis of azide-A-DSBSO (3) from pentaerythritol (5).
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Brønsted or Lewis acids in refluxing solvent, which required to produce 75% of pure 10, but the crude product was carried
isolation and recycling of a small amount of the recovered on in the sequence. By comparison to the original route, this
acetal 9. By carefully optimizing the catalyst quantity, time, method eliminates four steps in the sequence. The acetal syn-
and temperature in microwave reaction conditions the conver- thesis was carried out using the Noyori protocol,16 which was
sion to diol 10 could be pushed to completion with isolated found to be more reliable than the original acid-catalyzed
yields of ∼94%. Diol 10 is a key intermediate in the synthesis, method. Diol 10 was silylated and then combined with ketone
and it is the branch point for the preparation of the azide 11 in the presence of TMSOTf to give acetal 12 in 65% overall
cross-linker 3 and the alkyne cross-linker 4. It was prepared in yield. Hydrolysis of the dimethyl ester 12 used LiOH as before.
five steps following this route in 64% overall yield with only The bis-NHS ester 14 was prepared using in situ generated
one chromatographic purification.14
TFA-NHS,17 which lead to similar overall yields but shorter
The remainder of the synthesis of azide cross-linker 3 is reaction times, fewer side products, and a more reliable purifi-
outlined in Scheme 1. The diol 10 was reacted with 5-azidopen- cation. Finally, oxidation to the bis-sulfoxide as previously
tan-2-one (11)15 with acid catalysis using a Dean-Stark trap to described gave azide 3. The new route requires only six steps,
remove water. The acetal 12 was isolated in 77% yield by three chromatographic purifications, and led to an overall
chromatography. The NHS esters were introduced in two yield of 38%.14 It is more convenient and reliable than the
steps: hydrolysis of the methyl ester and coupling with prior route and has been used to produce multiple grams of
N-hydroxysuccinimide using EDC·HCl in dimethylformamide azide-A-DSBSO 3.
(DMF). The overall yield was typically around 60% and the
The 5-azidopentan-2-one was initially prepared by the alkyl-
bis-NHS ester 14 was isolated by chromatography. The final ation of commercially available bromide 18 with NaN3.15 The
oxidation was carried out with meta-chloroperoxybenzoic acid very high cost of bromide 18 led us to develop a more econo-
(m-CPBA); the oxidant was added in aliquots and conversion mical approach starting with lactone 17 (Scheme 3). The
was monitored by ESI-MS until the starting material and lactone 17 was treated with HBr to generate the required 5-bro-
monosulfoxide were no longer present. Azide cross-linker 3 mopentan-2-one (18). The standard displacement with sodium
was isolated by extraction. This route produced several azide gave the desired ketone 11 in good overall yield. Scaling
grams of the cross-linker 3 in ca. 18% overall yield for the up the synthesis of azide-A-DSBSO 3 required a significant
sequence.14
quantity of the volatile azide 11, and the starting with lactone
In the course of our in vivo studies,6 we found that the 17 was both effective and economical.
azide 3 crossed the membrane and produced cross-links in tar-
The alkyne 4 was prepared from diol 10 using a very similar
geted protein complexes.6 The studies required a large excess route (Scheme 4). The acetal 19 was formed from 5-hexyn-2-
of cross-linker, and led to an ongoing demand for more one and the diol under Dean-Stark conditions. The dimethyl
material. Although the original optimized synthesis in ester was hydrolyzed to a diacid using LiOH, and the di-NHS
Scheme 1 was effective, it did require nine steps. A shorter ester was prepared using TFA-NHS reagent.17 Di-NHS ester 20
route was developed that incorporated several improvements was isolated in 60% yield using this method. The same com-
in the individual transformations and avoided the use of pro- pound was also prepared using an EDCI coupling, but the
tecting groups. The new route is presented in Scheme 2.
yield was lower and the purification was more difficult. The
The new route begins with the commercially available and m-CPBA oxidation was conducted as described for the azide
inexpensive dibromide 15 and thiol 16. Direct alkylation with substrate to give the Alkyne-A-DSBSO 4. The route required
K2CO3 in DMF generated the key intermediate 10 in a single only four steps from diol 10 and made cross-linker 4 available
step. Diol 10 could be purified by chromatography on silica gel on gram scale.
Scheme 2 Improved synthesis of azide-A-DSBSO (3) beginning with 2,2-bis(bromomethyl)propane-1,3-diol (15).
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MSn analysis of azide-A-DSBSO cross-linked Ac-myelin peptide
Given the similarity of cleavable C–S bonds in azide-A-DSBSO
and DSSO, we anticipated that azide-A-DSBSO cross-linked
peptides would display comparable fragmentation character-
istics to DSSO cross-linked peptides (Fig. 3A).4 In such experi-
ments, MS2 produces peptide fragments that are modified
with remnant portions of the cross-linking reagents. These
remnants are not identical thus producing two products with a
separation of 254 Da, the αA and the αT fragments. The αA frag-
ment has an alkene group, while the other half of the cleaved
linker results in a terminal thiol group (after hydrolysis of the
sulfenic acid intermediate). Although azide-A-DSBSO contains
four C–S bonds due to the presence of two sulfoxide groups,
the two central C–S bonds cannot undergo fragmentation due
Scheme 3 Improved synthesis of 5-azidopentan-2-one (11).
Scheme 4 Synthesis of alkyne-A-DSBSO (4) from diol 10.
Fig. 3 MSn analysis of azide-A-DSBSO cross-linked Ac-myelin synthetic peptide. (A) Schematic illustration of MS2 analysis of a azide-A-DSBSO
interlinked homodimeric peptide (α–α). During collision-induced dissociation in MS2, cleavage of either of the two symmetric MS-cleavable C–S
bonds leads to physical separation of the two inter-linked peptides, thus generating pairs of peptide fragments: i.e. αA and αT. (B) The interlinked Ac-
myelin was detected as multiple charged ions (m/z 1001.823+, m/z 751.624+, m/z 601.455+, m/z 501.416+) in MS1. (C and D) MS2 spectra of interlinked
2+
Ac-myelin at two different charge states: (C) [α–α]4+ and (D) [α–α]6+. As shown, two predicted peptide fragment pairs were observed as αT2+/βA
(m/z 674.352+/819.892+) and αT3+/βA3+ (449.903+/546.933+) in (C) and (D) respectively.
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to the lack of β-hydrogens. Therefore, only the two C–S bonds
closer to cross-linked residues are expected to fragment during
MS2. To examine MS2 fragmentation patterns of azide-
A-DSBSO cross-linked peptides during MSn analysis, we have
first cross-linked and analyzed model peptide Ac-myelin. MS1
analysis detected azide-A-DSBSO cross-linked Ac-myelin (α–α)
homodimer at four different charge states (m/z 501.416+,
601.455+, 751.624+, 1001.823+) (Fig. 3B). MS2 analyses of inter-
linked Ac-myelin homodimer at different charge states yielded
the expected fragmentation of two identical inter-linked pep-
tides, i.e. a characteristic fragment pair (αA/αT). As an example,
the fragment pair αA/αT detected in MS/MS spectra of the
quadruply (m/z 751.624+) and sextuply (m/z 501.416+) charged
inter-linked Ac-myelin (α–α) was displayed in Fig. 3C and 3D,
respectively. The results demonstrate that the MS-cleavable
C–S bonds in azide-A-DSBSO cross-linked peptides are prefer-
entially fragmented during MS2 analysis prior to the breakage
of peptide backbones. Similar results were observed with
alkyne-A-DSBSO cross-linked Ac-myelin peptide (data not
shown) as azide-A-DSBSO and alkene-A-DSBSO are almost
identical in structures.
LC/MSn analysis of DSBSO cross-linked peptides of
cytochrome C after enrichment
To demonstrate the applicability of azide-A-DSBSO for XL-MS
studies, we have cross-linked model protein cytochrome C with
azide-A-DSBSO. Cytochrome C has been used extensively by us
and other groups for evaluating cross-linking reagents because
it is a small protein with a high number of lysine residues.
Given its success in the past for cross-linking studies,4,18 we
decided to use it as the model protein for characterizing our
new cross-linking reagent. The resulting cross-linked cyto-
chrome C products were conjugated with BARAC-biotin,19
affinity purified by binding to Streptavidin beads, and digested
with trypsin. The cross-linked peptides were eluted from the
beads with acid, and thus became acid-cleaved products of
azide-A-DSBSO cross-linked peptides, i.e. DSBSO cross-linked
peptides, which were then subjected to LC/MSn analysis. The
general workflow and the structure of the cross-linked peptides
leading up to LC/MSn analysis are illustrated in Fig. 4. As illus-
trated, the acid-cleaved products are the final analytes for
LC/MSn analysis. It is noted that the acid-cleaved products of
azide-A-DSBSO and alkyne-A-DSBSO cross-linked peptides are
the same, because the differentiated group is lost during the
acid elution of cross-linked peptides from affinity matrix
during enrichment. Since the cleavable C–S bonds in DSBSO
are similar to those in DSSO, the general data analysis work-
flow for the identification of DSBSO cross-linked peptides by
LC/MSn is similar to the analysis of DSSO cross-linked pep-
tides.4 There are three types of cross-linked peptides, i.e. dead-
end, intra-linked, and inter-linked peptides. Among them,
inter-linked peptides provide the most informative structural
details for defining protein–protein interaction interfaces.
Therefore, we are most interested in identifying inter-linked
peptides between the same and/or different proteins. As an
example, Fig. 5 describes a representative MSn analysis of a
Fig. 4 Work flow for affinity purification of cross-linked cytochrome C
proteins. The MS2 fragments resulting from CID cleavage sites are
shown.
DSBSO inter-linked cytochrome C peptide (α–β) that was
detected as a quadruply charged ion (m/z, 510.01664+). As
shown, MS2 analysis resulted in two pairs of peptide fragments
(i.e. αA/βT or αT/βA), characteristic fragmentation of inter-linked
heterodimeric peptide. Subsequent MS3 analysis of MS2 frag-
ment αA (m/z 430.752+) and βA (m/z 489.282+) determined their
sequences as KAYIPGTK and M(ox)IFAGIKAK respectively, in
which KA is modified with the alkene moiety. Integration of
MS1, MS2, and MS3 results has unambiguously determined
this DSBSO cross-linked cytochrome
C
peptide as
[74KYIPGTK80 inter-linked to 81M(ox)IFAGIKK88], in which a
cross-link was formed between K74 and K87 in cytochrome C.
In total, LC/MSn analysis of enriched cross-linked cyto-
chrome C identified 7 unique inter-linked peptides (Sup-
plemental Table 1†). In addition, 11 unique dead-end and 5
unique intra-linked cytochrome C peptides were identified
since all types of cross-linked peptides can be selectively
enriched (data not shown). The results are comparable to
those obtained using DSSO cross-linking,4 demonstrating the
effectiveness of azide-A-DSBSO based XL-MS strategy. Although
it is not necessary to enrich cross-linked peptides for simple
proteins like cytochrome C, it is evident that such a process is
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Fig. 5 MSn analysis of a representative DSBSO 3 inter-linked peptide (α–β) of cytochrome C (m/z 510.024+). (A) MS1 and (B) MS2 spectra of the
selected peptide. In MS2 spectrum, two pairs of peptide fragments: i.e. αA/βT (m/z 430.752+/580.282+) and (αT/βA(521.752+/489.282+) were detected.
Note: αA, βA, and KA are alkene modified species (+54 Da); αT and βT are unsaturated thiol modified species (+236 Da). (C and D) Respective MS3
spectra of αA (m/z 430.752+) and βA (489.282+) fragments detected in MS2. The detection of a series of y and b ions has unambiguously identified
their sequences as KAYIPGTK and MoxIFAGIKAK respectively. Mox: oxidized methionine.
essential for mapping protein interaction interfaces at the
Abbreviations
systems level.6
Ac
Acetate
Alkyne-
A-DSBSO
Azide-
A-DSBSO
BARAC
CID
Alkyne-tagged, acid-cleavable disuccinimidyl-
bissulfoxide
Azide-tagged, acid-cleavable disuccinimidyl-
bissulfoxide
Biarylazacyclooctynone
Collision-induced dissociation
Camphorsulfonic acid
Copper(I) catalyzed azide–alkyne cycloaddition
N,N-Diisopropylethylamine
Dimethylformamide
Disuccinimidylsulfoxide
N-(3-Dimethylaminopropyl)-N′-ethylcarbodii-
mide hydrochloride
Conclusion
Full experimental details are presented for the preparation of
two CID-cleavable lysine cross-linkers, an azide (azide-
A-DSBSO) and an alkyne (alkyne-A-DSBSO), along with initial
characterization using a model peptide and a model protein.
The syntheses are not trivial, but the optimized procedures
reported herein make these useful compounds available on
multigram scale. The azide and alkyne functional groups are
suitable for click enrichment strategies. The cross-linkers
described here have been utilized in mammalian HEK-293
cells6 and to facilitate the study of the interaction of subunits
in the proteasome complex, which is responsible for degra-
dation of ubiquitin tagged proteins.6 The importance of devel-
oping XL-MS reagents that are applicable for in vivo studies is
significant because protein–protein interactions are involved
in most cell function and are not well understood. We hope
that this report detailing the synthesis of these cross-linking
agents will enable others to prepare and utilize them to study
protein–protein interactions.
CSA
CuAAC
DIPEA
DMF
DSSO
EDC·HCl
Et3N
HB
ESI-MS
In(OTf)3
KAYIPGTK
Triethylamine
His-bio
Electrospray ionization-mass spectrometry
Indium(III) trifluoromethanesulfonate
Lysine(alkene
modified)-tyrosine-isoleucine-
proline-glycine-threonine-lysine peptide
Liquid chromatography-mass spectrometry
Methanol
Methionine(oxidized)-isoleucine-phenylalanine-
alanine-glycine-isoleucine-lysine(alkene modi-
fied)-lysine
LC/MS
MeOH
M(ox)
IFAGIKAK
Methods
See ESI† for experimental details for the synthesis of each m-CPBA
meta-Chloroperoxybenzoic acid
Methanesulfonyl chloride
cross-linking reagent.
MsCl
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5 A. Leitner, T. Walzthoeni, A. Kahraman, F. Herzog,
O. Rinner, M. Beck and R. Aebersold, Mol. Cell. Proteomics,
2010, 9, 1634–1649.
NHS
Ph-H
Pyr.
N-Hydroxysuccinimidyl
Benzene
Pyridine
6 R. M. Kaake, X. Wang, A. Burke, C. Yu, W. Kandur, Y. Yang,
E. J. Novitsky, T. Second, J. Duan, A. Kao, S. Guan,
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Proteomics, 2014, 13, 3533–3543, DOI: 10.1074/mcp.
M114.042630.
QTAX
Quantitative analysis of tandem affinity purified
in vivo cross-linked (X) protein complexes
Trifluoroacetic anhydride
N-Trifluoroacetoxy succinimide
Tetrahydrofuran
TFAA
TFA-NHS
THF
7 D. Paramelle, G. Miralles, G. Subra and J. Martinez,
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TMSCl
TMSOTf
TsOH
XL-MS
μw
Trimethylsilyl chloride
Trimethylsilyl trifluoromethanesulfonate
para-Toluenesulfonic acid
Cross-linking mass spectrometry
Microwave
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This work was supported by National Institutes of Health
grants RO1GM074830-06A1 and R21CA161807 to L.H., and
Biomedical Informatics Training predoctoral fellowship to A.K.
(5T15LM007443-10 to Pierre Baldi). Eric J. Novitsky was sup-
ported by an Institutional Chemical and Structural Biology
Training Grant predoctoral fellowship (T32-GM10856).
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