Developing new isotope-coded mass spectrometry-cleavable cross-linkers for elucidating protein structures

Article abstract of DOI:10.1021/ac403636b

Structural characterization of protein complexes is essential for the understanding of their function and regulation. However, it remains challenging due to limitations in existing tools. With recent technological improvements, cross-linking mass spectrometry (XL-MS) has become a powerful strategy to define protein-protein interactions and elucidate structural topologies of protein complexes. To further advance XL-MS studies, we present here the development of new isotope-coded MS-cleavable homobifunctional cross-linkers: d₀- and d₁₀-labeled dimethyl disuccinimidyl sulfoxide (DMDSSO). Detailed characterization of DMDSSO cross-linked peptides further demonstrates that sulfoxide-containing MS-cleavable cross-linkers offer robust and predictable MS2 fragmentation of cross-linked peptides, permitting subsequent MS3 analysis for simplified, unambiguous identification. Concurrent usage of these reagents provides a characteristic doublet pattern of DMDSSO cross-linked peptides, thus aiding in the confidence of cross-link identification by MSⁿ analysis. More importantly, the unique isotopic profile permits quantitative analysis of cross-linked peptides and therefore expands the capability of XL-MS strategies to analyze both static and dynamic protein interactions. Together, our work has established a new XL-MS workflow for future studies toward the understanding of structural dynamics of protein complexes.

Full text of DOI:10.1021/ac403636b

Article
pubs.acs.org/ac
Open Access on 01/28/2015
Developing New Isotope-Coded Mass Spectrometry-Cleavable Cross-
Linkers for Elucidating Protein Structures
†
‡
†
‡
,†
Clinton Yu, Wynne Kandur, Athit Kao, Scott Rychnovsky, and Lan Huang*
†Department of Physiology and Biophysics, University of California, Medical Science I, D233, Irvine, California 92697, United States
‡Department of Chemistry, University of California, Irvine, California 92697, United States
*
S Supporting Information
ABSTRACT: Structural characterization of protein complexes is
essential for the understanding of their function and regulation.
However, it remains challenging due to limitations in existing tools.
With recent technological improvements, cross-linking mass spec-
trometry (XL-MS) has become a powerful strategy to define protein−
protein interactions and elucidate structural topologies of protein
complexes. To further advance XL-MS studies, we present here the
development of new isotope-coded MS-cleavable homobifunctional
cross-linkers: d - and d -labeled dimethyl disuccinimidyl sulfoxide
0
10
(
DMDSSO). Detailed characterization of DMDSSO cross-linked
peptides further demonstrates that sulfoxide-containing MS-cleavable
cross-linkers offer robust and predictable MS2 fragmentation of cross-
linked peptides, permitting subsequent MS3 analysis for simplified, unambiguous identification. Concurrent usage of these
reagents provides a characteristic doublet pattern of DMDSSO cross-linked peptides, thus aiding in the confidence of cross-link
n
identification by MS analysis. More importantly, the unique isotopic profile permits quantitative analysis of cross-linked peptides
and therefore expands the capability of XL-MS strategies to analyze both static and dynamic protein interactions. Together, our
work has established a new XL-MS workflow for future studies toward the understanding of structural dynamics of protein
complexes.
rotein complexes represent essential functional entities in
cells for carrying out multiple biological processes
between cross-linked residues can be converted to distance
restraints for protein homology modeling.
6
P
including translation, replication, cell division, and cell cycle
control. Protein−protein interactions are integral in modulating
the assembly, structure, and function of protein complexes.
Perturbations of endogenous protein−protein interactions can
result in deleterious effects on cellular activities and lead to
human disease. In recent years, protein−protein interaction
interfaces have become a new and attractive platform for
The major challenges in XL-MS studies are the detection of
low-abundance cross-linked peptides and their unambiguous
identification. The complexity in peptide mixtures often
impedes MS detection of cross-linked peptides due to the
presence of significantly more abundant noncross-linked
peptides. In addition, heterogeneous populations of cross-
linked products, i.e., interlinked, intralinked, and dead-end
modified peptides further complicates the analysis. To facilitate
the detection of cross-linked peptides, one strategy is to
selectively enrich cross-linked products for MS analysis using
enrichable cross-linkers containing either an affinity tag (e.g.,
1
therapeutics. Therefore, characterization of structures and
interaction dynamics of protein complexes is critical to
understanding their function and regulation, thus unraveling
molecular mechanisms underlying human pathologies and
providing insight on potential targets for drug development.
Traditional structural tools such as nuclear magnetic resonance
1
0,11
biotin tag)
or a chemical handle that allows subsequent
12
addition of an affinity tag through chemical conjugation.
Another strategy is to incorporate stable isotopes in cross-
(
NMR) and X-ray crystallography are able to yield detailed,
linked peptides to generate characteristic isotopic profiles, thus
high-resolution information on protein structures. However,
these technologies have difficulty in analyzing heterogeneous
and dynamic protein complexes. Following decades of method
development alongside technological advances in mass
spectrometry, cross-linking mass spectrometry (XL-MS) has
emerged as a powerful strategy not only for mapping protein
interaction networks
protein complexes.
used to derive topological ordering of protein complexes by
computational modeling.
6
,7,10,13−16
separating them from noncross-linked peptides.
This
differentiation can be achieved by first carrying out enzymatic
16
18
digestion of cross-linked proteins in O and 1 O water,
3
respectively, and then mixing prior to MS analysis. Although
effective, enzymatic incorporation of 18O is troublesome as its
2
−4
but also for structural elucidation of
The cross-links between proteins can be
5
−8
Received: November 7, 2013
Accepted: January 28, 2014
Published: January 28, 2014
8
,9
In addition, spatial distances
©
2014 American Chemical Society
2099
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Figure 1. Chemical synthesis schemes for (A) d -DMDSSO and (B) d -DMDSSO.
0
10
labeling efficiency relies heavily on peptide sequences.
and has been successfully applied to elucidate structures of
proteasome complexes.
To further advance XL-MS studies of protein complexes, we
have developed a pair of new isotope-coded DSSO derivatives,
18
8,12
Interestingly, performing protein cross-linking in O water
1
8
can result in the incorporation of one O to dead-end modified
peptides but not to other types of peptides, thus effectively
10
distinguishing them from intralinked and interlinked peptides.
i.e., d - and d10-labeled dimethyl-disuccinimidyl sulfoxide
0
However, a common practice to produce cross-linked peptides
as isotopic pairs for easy identification is to cross-link proteins
with a 1:1 mixture of nonlabeled and labeled cross-link-
(DMDSSO). Incorporation of deuterium labeling into our
robust sulfoxide-containing MS-cleavable cross-linker adds new
features that not only enhance the detection and identification
of cross-linked peptides but also provide the capability of
quantifying cross-linked peptides. Here we present the detailed
characterization of DMDSSO-based cross-linking strategy using
6
,7,14−17
ers.
Apart from the detection of cross-linked peptides,
unambiguous identification of interlinked peptides by peptide
sequencing is challenging when noncleavable cross-linkers are
used. This is due to the difficulty in interpreting convoluted
tandem mass spectra resulted from the fragmentation of two
interlinked peptides. Despite recent innovation in bioinfor-
matics tools that have been developed to better dissect
synthetic peptides and model protein cytochrome C. We have
n
compared MS analyses of d - and d -DMDSSO cross-linked
0
10
peptides and performed quantitative assessments of cross-
linked peptides with different sample preparation strategies.
1
8−22
fragmentation data of interlinked peptides,
further
EXPERIMENTAL PROCEDURES
■
improvements are required to make it as generally applicable
as that for identifying single peptide sequences. To circumvent
these problems, various types of cleavable cross-linkers, e.g.,
MS-, photo-, and chemical-cleavable reagents, have been
developed to facilitate MS identification of cross-linked
peptides. Among them, MS-cleavable reagents appear to be
Materials and Reagents. General chemicals were
purchased from Fisher Scientific or VWR International, bovine
heart cytochrome C (98% purity) from Sigma-Aldrich, and Ac-
Myelin peptide (Ac-ASQKRPSQRHG, 92.7% purity) from
American Peptide (Sunnyvale, CA).
Synthesis and Characterization of d -DMDSSO and
0
1
1,12,17,23,24
most attractive for XL-MS studies,
owing to their
d -DMDSSO. The synthesis of DMDSSO was depicted in
1
0
unique capability of fragmenting cross-links during collision-
induced dissociation (CID) and thus facilitating subsequent
peptide sequencing for unambiguous identification. Recently,
we have developed a novel MS-cleavable homobifunctional
NHS ester, disuccinimidyl sulfoxide (DSSO), in which the MS-
cleavable C−S bond cleaves preferentially during MS2 analysis
prior to the breakage of the peptide backbone. This cleavage
permits robust, reliable, and characteristic CID-induced
fragmentation of cross-linked peptides unique to their cross-
linking types, generating distinct MS2 fragment ions for
subsequent MS3 sequencing. This novel integrated workflow
has proven to be effective for fast and accurate identification of
cross-linked peptides using conventional bioinformatics tools
Figure 1. Briefly, the preparation of d -DMDSSO began with
0
addition of thioacetic acid to methyl methacrylate. Methanol
and triethylamine were added to the mixture along with
another equivalent of methyl methacrylate to afford the
symmetrical diester in one pot. The diester was hydrolyzed
with lithium hydroxide in THF/H O before coupling with
2
12
NHS, in the presence of trifluoroacetic anhydride, pyridine, and
25
DMF. Lastly, oxidation of the sulfide to the sulfoxide yielded
12
the desired linker as described. The preparation of d -
1
0
DMDSSO was carried out similarly, beginning with commer-
cially available d -methyl methacrylate. The details of the
8
chemical characterization are described in the Supporting
Information.
2
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Figure 2. Characteristic MS2 fragmentation patterns for DMDSSO cross-linked peptides. MS2 fragmentation of (A) d -DMDSSO interlinked
0
heterodimer α−β. (B) d -DMDSSO intralinked peptide αintra. (C) Dead-end modified peptide αDN. (D) The conversion scheme of α to α . (E)
0
S
T
Illustrations of α , α , and α fragments with lysines modified with d -DMDSSO remnants.
A*
S*
T*
10
Cross-Linking of Synthetic Peptides with d - and d -
as the enzyme with four maximum missed cleavages allowed.
Protein N-terminal acetylation, methionine oxidation, N-
terminal conversion of glutamine to pyroglutamic acid,
asparagine deamidation, and cysteine carbamidomethylation
were selected as variable modifications. In addition, three
defined modifications on uncleaved lysines and free protein N-
0
10
DMDSSO. Synthetic peptide Ac-Myelin was dissolved in
DMSO to 1 mM and cross-linked with either d - or d -
0
10
DMDSSO in a 1:1 molar ratio of peptide to cross-linker in the
presence of 1 equiv of diisopropylethylamine. The resulting
samples were diluted to 5 pmol/μL in 3% ACN/2% formic acid
for MS analysis.
termini were selected: alkene (A, C
O, + 73 Da), sulfenic acid (S, C
S, + 123 Da), and unsaturated thiol (T, C
100 Da; or T*, C H D OS, + 105 Da) modification, due to
H
O, + 68 Da; or A*,
S, + 118 Da; or
OS,
4
4
Cross-Linking of Cytochrome C with d - and d -
C
S*, C
+
4
H
−1
D
5
D
H O
4 6 2
0
10
DMDSSO. A volume of 40 μL of 200 μM cytochrome C in
PBS buffer (pH 7.4) was reacted with d - or d -DMDSSO in a
4
H
1
5
O
2
H
4 4
4
−1
5
0
10
remnant moieties for d - (i.e., A, S, T) or d -DMSSO (i.e., A*,
molar ratio of 1:10 (protein−cross-linker) for 2 h at room
temperature and quenched with excess ammonium bicarbonate.
Samples were then subjected to SDS-PAGE and visualized by
Coomassie blue. The dimerized bands were excised, reduced
with TCEP for 30 min, and alkylated with chloroacetamide for
0
10
S*, T*) cross-linker, respectively. Initial acceptance criteria for
peptide identification required a reported expectation value
≤
0.1.
MS-Bridge was used to confirm the identification of cross-
linked peptides by mass mapping against bovine cytochrome C
4
5 min in the dark, and then digested with trypsin at 37 °C
12
with the parent mass error set as ±10 ppm. The in-house
program Link-Hunter is a revised version of the previously
written Link-Finder program, designed to automatically validate
overnight. Peptide digests were extracted, concentrated, and
reconstituted in 3% ACN/2% formic acid for MS analysis.
Liquid Chromatography−Multistage Tandem Mass
n
n
and summarize cross-linked peptides based on MS data and
Spectromtery (LC MS ). DMDSSO cross-linked peptides
8
,12
n
database searching results as previously described.
were analyzed by LC−MS utilizing an LTQ-Orbitrap XL MS
(
ThermoFisher, San Jose, CA) coupled online with an Eksigent
12
RESULTS AND DISCUSSION
NanoLC system (Dublin, CA) as previously described. Each
■
n
MS experiment has a duty cycle of 1.3 s, consisting of one MS
Design and Synthesis of New Isotope-Coded DSSO
Derivatives. In order to further facilitate MS identification of
cross-linked peptides and allow quantitative determination of
structural dynamics of protein complexes, we aimed to generate
deuterium labeled MS-cleavable cross-linkers. Given our
previous success of DSSO-based XL-MS strategies in protein
scan in FT mode (350−1400 m/z, resolution of 60 000 at m/z
00) followed by two data-dependent MS2 scans in FT mode
resolution of 7500) with normalized collision energy at 15%
on the top two MS peaks with charges at 3+ or up, and three
MS3 scans in the LTQ with normalized collision energy at 35%
on the top three peaks from each MS2.
4
(
8
,12
structural characterization, we first attempted to produce d4-
DSSO by introducing deuterium at the positions alpha to the
carbonyls through deuterium exchange (Supplementary Figure
1 in the Supporting Information). Although feasible, complete
labeling was problematic due to slow exchange. Additionally,
labeling with four deuteriums proved to be insufficient for
effective separation of highly charged d /d -DSSO cross-linked
Data Analysis of Cross-Linked Peptides. Data process-
n
12
ing of LC−MS spectra was carried out as described. MS3
data was subjected to a developmental version of Protein
Prospector (v. 5.10.10) for database searching, using Batch-Tag
against cytochrome C sequence (SwissProt accession no.
P62894) with mass tolerances for parent ions and fragment
ions set as ±20 ppm and 0.6 Da, respectively. Trypsin was set
0
4
n
peptide pairs (4+ and above) during MS analysis. Therefore,
2
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n
Figure 3. MS analyses of d - and d -DMDSSO interlinked Ac-Myelin peptides. (A) MS spectrum of d -interlinked Ac-Myelin. (B−D) MS2 spectra
0
10
0
6+
5+
4+
of d -interlinked Ac-Myelin at three different charge states: (B) [α−α] , (C) [α−α] , and (D) [α−α] . (E) MS spectrum of d -interlinked Ac-
Myelin. (F−H) MS2 spectra of d -interlinked Ac-Myelin at three different charge states: (F) [α−α] , (G) [α−α] , and (H) [α−α] .
0
10
6+
5+
4+
10
d8-labeled DSSO would be ideal; however, incorporation of
eight deuteriums in DSSO appeared to be less practical due to
cost and experimental difficulties. To circumvent this problem,
we have designed a new derivative of DSSO, dimethyl
disuccinimidyl sulfoxide (DMDSSO). With the commercial
intralinked peptide αintra, one peptide fragment (i.e., αA+S) is
anticipated, carrying an alkene- and a sulfenic acid-modified
lysine, respectively (Figure 2B). This MS2 fragment ion αA+S
actually represents two different ion species that have identical
peptide sequences and m/z values but transposed DMDSSO
availability of methyl methacrylate and d -methyl methacrylate,
remnant-modified lysine residues. For a d -DMDSSO dead-end
8
0
the synthesis of d - or d -DMDSSO is economical and
modified peptide (αDN), two peptide fragments (i.e., α and α )
0
10
A
S
straightforward (Figure 1). Similar to DSSO, DMDSSO also
has an ideal length (an average extended length of 9.3 Å) for
structural proteomics studies.
are expected (Figure 2C). It is noted that the sulfenic acid
moiety often undergoes dehydration to become a more stable
and dominant unsaturated thiol moiety (i.e., T, + 100 Da) as
12
Expected CID Fragmentation Patterns of d - and d -
previously described (Figure 2D). This conversion does not
appear to complicate data analysis as observed for DSSO cross-
0
10
DMDSSO Cross-linked Peptides. Three types of cross-
linked products can result from the digestion of cross-linked
proteins: interlinked, intralinked, and dead-end modified
peptides. Previously we have shown that DSSO cross-linked
peptides display characteristic fragmentation patterns during
MS2 analysis due to preferential cleavage of CID-cleavable C−
12
linked peptides. In comparison to d -DMDSSO cross-linked
0
peptides, fragmentation patterns of d -DMDSSO cross-linked
10
peptides should be the same except all of the d -DMDSSO
1
0
remnants (i.e., A*, alkene; S*, sulfenic acid; or T*, unsaturated
thiol) are 5 Da higher in mass due to the presence of 5
deuteriums after cleaving the C−S bond (Figure 2E). In
addition to distinct MS2 fragmentation patterns, DMDSSO
cross-linked peptides have fixed mass relationships between
parent ions and their respective fragment ions, similar to those
12
S bonds adjacent to the sulfoxide. Aside from two additional
methyl groups, DMDSSO has a structure very similar to DSSO,
with two symmetric MS-cleavable C−S bonds. Therefore, we
expect that DMDSSO cross-linked peptides will display the
same characteristic MS2 fragmentation patterns as DSSO cross-
linked peptides. Since deuterium labeling should not interfere
with peptide fragmentation, d - and d -DMDSSO cross-linked
12
of DSSO cross-linked peptides, thus providing an additional
confirmation of the identified cross-linked peptides at the MS2
level. Together with MS3 sequencing and MS1 mass matching,
three different types of evidence can be obtained for the
identification of DMDSSO cross-linked peptides with signifi-
cantly improved confidence and accuracy.
0
10
n
peptides would behave similarly during MS analysis. For
simplicity, we use d -DMDSSO cross-linked peptides to
0
illustrate their predicted fragmentation patterns (Figure 2).
Prior to peptide backbone fragmentation, MS2 analysis
selectively cleaves either of the two symmetric C−S bonds in
the linker region of DMDSSO cross-linked peptides, yielding
peptide fragments with predictable modifications (due to the
remnants of DMDSSO) on cross-linked lysine residues. For a
Characterization of DMDSSO Cross-Linked Model Pep-
n
tides by MS Analysis. We first performed DMDSSO cross-
linking on synthetic peptide Ac-Myelin. Under our exper-
imental conditions, the resulting cross-linked products were
primarily interlinked Ac-Myelin homodimer (α−α), which
d -DMDSSO interlinked peptide α−β, cleavage of a C−S bond
were detected as a series of multiply charged ions for d -
0
0
6
+
5+
4+
leads to physical separation of the two interlinked peptides into
DMDSSO (m/z 462.9033 , 555.2822 , 693.8497 ) and d -
10
6
+
5+
4+
a pair of peptide fragments (i.e., α /β or α /β ), in which α
DMDSSO (m/z 464.5796 , 557.2951 , 696.3656 ), respec-
tively (Figure 3A,E). There is a 10 Da mass difference between
d - and d -labeled cross-linked peptides due to incorporation
A
S
S
A
and β peptide fragments are modified by two complementary
cross-linker remnant moieties, i.e., alkene (A) and sulfenic acid
0
10
(
S) (Figure 2A). Thus, the resulting MS2 peptide fragments
of 10 deuteriums in d -DMDSSO. As shown in Figure 3B,
10
can be subjected to MS3 sequencing for unambiguous
MS2 analysis of the sextuply charged d -interlinked Ac-Myelin
0
12
6+
identification of interlinked peptides. For a d -DMDSSO
(d , α−α ) yielded a pair of dominant fragment ions (α /α ),
0
0
A
T
2
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Figure 4. General workflow for the analysis and identification of d /d10 DMDSSO cross-linked cytochrome C peptides.
0
n
Figure 5. MS analysis of d /d -DMDSSO interlinked cytochrome C peptides. (A) MS2 spectrum of a d -interlinked cytochrome C peptide α−β
0
10
0
3+
2+
(
m/z 574.6436 ). MS3 spectra of its MS2 fragment ions (B) α (m/z 415.76 ) and (C) β (m/z 874.40). (D) MS2 spectrum of a corresponding
d -interlinked cytochrome C peptide α−β (m/z 577.9993 ). MS3 spectra of its fragment ions (E) α (m/z 418.28 ) and (F) β (m/z 879.43).
A
T
3+
2+
10
A*
T*
demonstrating effective separation of the interlinked homo-
by MS3. Taken together, the results have proven that addition
of methyl substituents in the linker region does not change the
unique fragmentation of sulfoxide-containing MS-cleavable
cross-linked peptides, and the preferential cleavage of C−S
dimer as expected. Similarly, the α /αT* ion pair was also
A*
detected as the most abundant ions in MS2 spectrum for d -
1
0
6
+
interlinked Ac-Myelin peptide (d , α−α ) (Figure 3F),
1
0
n
indicating no interference from deuterium labeling. MS2
analyses of quadruply- and quintuply-charged Ac-Myelin
peptides also resulted in one pair of fragment ions (d , α /
bonds is independent of peptide charges. Thus, MS analysis of
DMDSSO cross-linked peptides can be performed the same
12
way as that of DSSO cross-linked peptides.
0
A
α ; d , α /α ) (Figure 3C,D,G,H), in which α or α
Characterization of DMDSSO Cross-Linked Cytochrome C
S
10
A* S*
S
S*
n
appears to be more dominant than α or α , respectively, in
by MS Analysis. We next evaluated the applicability of d - and
T
T*
0
contrast to the fragmentation of sextuply charged interlinked
peptides (Figure 3B,F). This observation may be due to the
susceptibility of highly charged species to fragmentation when
the same energy is applied to all precursor ions during CID
analysis regardless of their charge. Such fragmentation behavior
was previously observed for DSSO interlinked Ac-Myelin
d -DMDSSO for protein cross-linking. Model protein
1
0
cytochrome C has been extensively used to test various new
cross-linking strategies due to the large number of lysine
1
2,26
residues relative to its size.
In this work, DMDSSO cross-
linked cytochrome C was separated by 1-D SDS-PAGE and
visualized by Coomassie blue staining. In comparison to DSSO,
d - and d -DMDSSO showed comparable efficiency in protein
12
peptides as well. MS3 sequencing of α , α , α , and α
A
T
A*
T*
0
10
fragment ions confirmed the peptide sequences of d - and d -
cross-linking (Supplementary Figure 3 in the Supporting
Information). The general workflow for analyzing cross-linked
cytochrome C is illustrated in Figure 4. As shown, we first
analyzed in-gel digests of d - and d -DMDSSO dimerized
0
10
interlinked Ac-Myelin peptides unambiguously (Supplementary
Figure 2 in the Supporting Information), and none of the
DMDSSO remnants appear to complicate peptide sequencing
0
10
2
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cytochrome C separately. Figure 5A,D displays the respective
display the expected isotopic doublets with a 10 Da mass
difference, indicative of cross-linked peptides containing one
cross-link. This can be exemplified by respective peptide pairs
detected in MS1 for the three representative DMDSSO cross-
linked cytochrome C peptides described above (Supplementary
Figure 5A−C in the Supporting Information). Similar isotopic
signatures can also be detected in MS2 if d - and d -DMDSSO
MS2 spectra of a selected pair of d - and d -DMDSSO
0
10
3
+
interlinked cytochrome C peptides (m/z 574.6436 ,
77.9993 ), in which two pairs of peptide fragment ions (d ,
3+
5
0
α /β and α /β ; d , α /β and α /β ) were detected,
A
T
T
A
10
A* T*
T* A*
demonstrating characteristic fragmentation pattern of inter-
linked heterodimeric peptides. The most dominant fragment
pair ions, α /β for d - and α /β for d -labeled interlinked
0
10
cross-linked peptide pairs can be selected for CID analysis at
the same time or their respective MS2 spectra can be merged
together. The resulting MS2 isotopic doublets would have a
mass difference of 5 Da because MS2 fragments of DMDSSO
cross-linked peptides only carry five residual isotopic labels
(Figure 2E). Similarly, the unique MS2 isotopic signature can
be used to facilitate the identification and quantitation of cross-
linked peptides; however, special software is needed for
effective data analysis. Although quantitation at the MS1 level
is often preferred due to sensitivity, the detection of multiple
MS2 isotopic pairs can provide better statistics in quantitation.
In total, 33 unique interlinked cytochrome C peptides were
A
T
0
A* T*
10
peptides, were subsequently subjected to MS3 analysis (Figure
BC,E,F). On the basis of the series of y and b ions detected,
5
2
+
2+
the sequences of α (m/z 415.76 ) and α (m/z, 418.28 )
A
A*
were determined as K IFQVK and K IFQVK, respectively, in
A
A*
which the N-terminal K is modified with the alkene moiety.
Similarly, MS3 analysis of the corresponding β (m/z 874.40)
T
and βT* (m/z 879.43) identified their sequences as Ac-
GDVEK GK and Ac-GDVEK GK, respectively, where the K
T
T*
at the fifth position from N-terminus is modified with the thiol
moiety. Together with mass mapping of the parent ions using
MS-Bridge, the interlinked peptides were unambiguously
1
7
8
13
n
determined as [Ac- GDVEKGK interlinked to KIFQVK ],
in which a cross-link was formed between K5 and K8 in
cytochrome C.
identified, and 19 of them were identified based on MS
analysis of both d - and d -DMDSSO-cross-linked peptides
0
10
(Supplementary Table 1 and Supplementary Figure 7 in the
Supporting Information). The remaining 14 interlinks were
In addition to interlinked peptides, we have also identified
DMDSSO intralinked and dead-end modified cytochrome C
peptides, and their MS2 fragmentation patterns are the same as
depicted in Figure 2. For example, MS2 analysis of a selected
n
determined only by MS sequencing of either d - or d -
0
10
DMDSSO-cross-linked peptides. Importantly, the detection of
d0/d10 peptide doublets confirms the existence of the same
cross-linked peptides formed by both cross-linkers even if only
3
+
d -intralinked cytochrome C peptide (m/z 621.3203 ) yielded
0
3
+
n
a single dominant fragment ion (αA+T, m/z 615.32 )
Supplementary Figure 4A in the Supporting Information).
Similarly, its corresponding d -labeled cross-linked peptide
one of the d and d forms is analyzed by MS . These results
0
10
(
demonstrate that isotope-coded cross-linkers further improve
the identification of cross-linked peptides. The 33 identified
interlinked peptides represent 26 unique K−K linkages in
cytochrome C, C −C distances of which range from 5.3 to
1
0
3+
(
m/z 624.6746 ) also generated the same type of MS2
3
+
fragment ion (αA*+T*, m/z 618.67 ) (Supplementary Figure 4B
in the Supporting Information), corroborating well with the
predicted fragmentation unique to intralinked peptides. As for
dead-end modified peptides, they are expected to generate two
distinct MS2 fragment ions (Figure 2C). Such characteristic
fragmentation was observed for DMDSSO dead-end peptides
α
α
26.2 Å based on the reported monomer crystal structure (PDB
2B4Z). These distances are well within the expected range of
our cross-linkers (≤26 Å). However, it is noted that some of
the identified cross-linked peptides more likely represent
interprotein interlinks and may have larger spatial distances as
the dimerized cytochrome C was analyzed here. For example,
as demonstrated by MS2 spectra of a selected pair of d - (m/z
0
3+
3+
39
53
5
46.6116 ) and d -dead-end (m/z 549.9661 ) modified
the peptide [ KTGQAPGFSYTDANK ] was determined to
1
0
3
9
cytochrome C peptides, in which a pair of fragment ions αA/
be interlinked with another peptide [ KTGQAPGFSYTD-
55
α and α /α were detected, respectively (Supplementary
ANKNK ] through K39 to K53 linkage (Supplementary Table
1 in the Supporting Information). Interestingly, these two
interlinked peptides share a significant overlap in sequences,
strongly suggesting an interprotein interlink between a
cytochrome C dimer.
T
A* T*
Figure 4C,D in the Supporting Information). Taken together,
the results further demonstrate that DMDSSO cross-linked
peptides indeed produce specific MS2 fragmentation patterns
that are predictable and reliable for the determination of their
cross-link types, which allows subsequent MS3 analysis of
unique MS2 fragments for unambiguous identification of cross-
linked peptides. These features are consistent with those of
Previously, we have identified 14 interlinked cytochrome C
12
peptides using DSSO cross-linking, 8 of which have also been
determined by d /d -DMDSSO cross-linking in this study.
0
10
12
DSSO cross-linked peptides, further attesting the power and
general applicability of sulfoxide-containing MS-cleavable cross-
linkers in XL-MS studies.
Although each study has resulted in several unique cross-linked
peptides, it is noted that many of the identified interlinked
lysines are located in very close proximity within the sequence
of cytochrome C. For example, while K53 to K79 (11.6 Å)
linkage was found with DSSO cross-linking, K55 to K73 (11.6
Å) was only identified by DMDSSO cross-linking. Because of
the similar calculated distances within these cross-linked lysine
residues and the closeness of K53 to K55 as well as the
proximity between K73 and K79, we consider their interaction
regions are similar. Therefore, we clustered 17 lysines of
cytochrome C into 8 “groups”, in which adjacent lysines are
within a string of 6 amino acids (Supplementary Figure 6 in the
Supporting Information). In comparison to the interlinks
identified within these lysine groups, this work has mapped all
Detection of d /d -DMDSSO Cross-Linked Peptide
0
10
Pairs. In order to further facilitate the detection and
identification of cross-linked peptides, we next mixed the
digests of d - and d -DMDSSO cross-linked cytochrome C at
0
10
n
1
:1 for LC−MS analysis. When analyzed together, d - and d -
0
10
DMDSSO cross-linked peptides should be detected as isotopic
doublets in MS1 with defined mass differences (Δ(d − d ) =
10
0
n × 10 Da) depending on the number of cross-links (n) in a
given cross-linked peptide. In contrast, noncross-linked
peptides should be detected only as singlets. This provides
additional confirmation to cross-linked peptides identified by
n
12
MS . Not surprisingly, all of the cross-linked peptides identified
of the interlinked regions determined by DSSO cross-linking.
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Analytical Chemistry
Article
Figure 6. Quantitative analysis of d /d -DMDSSO cross-linked cytochrome C peptides. (A−E) Extracted ion chromatogram (XIC) overlays for a
0
10
3
+
3+
selected d - and d -interlinked peptide pairs (m/z 574.64 /578.00 ) when the digests of d - and d -DMDSSO cross-linked peptides were mixed in
0
10
0
10
the ratio of 5:1, 2:1, 1:1, 1:2, and 1:5, respectively. The shaded areas represent the XICs of d -interlinked peptides. (F−J) Representative MS spectra
10
obtained for each corresponding overlaid XICs shown in parts A−E. (K) Observed ratios of d /d ion signals for the 5 selected interlinked
0
10
cytochrome C peptides. Their sequences are shown in the inset.
In addition, 5 additional ones derived from 10 DMDSSO cross-
linked peptides were identified, representing the most extensive
cross-linking data on cytochrome C. These results are more
likely attributed to combined improvements in sample
preparation, data acquisition, and usage of two isotope-coded
cross-linkers separately and simultaneously.
cross-linked cytochrome C with a 1:1 mixture of d - and d -
0 10
DMDSSO and then analyzed the resulting cross-linked peptide
digests by MS (Supplementary Figure 5D−F in the Supporting
Information). In comparison, corresponding d - and d -labeled
0
10
cross-linked peptides display similar relative abundance ratios
regardless of whether mixing was done before or after protein
cross-linking. These results suggest that our isotopically labeled
cross-linkers are indeed comparable in their ability to cross-link
proteins and that the resulting d - and d -labeled cross-linked
In contrast to previous analysis of the entire cross-linked
cytochrome C mixture in which the monomeric form was the
12
most abundant species, here we have only focused on
analyzing gel-separated cytochrome C dimer bands to decrease
sample complexity. Because most noncross-linked tryptic
peptides, dead-end modified, and intralinked cross-linked
peptides have lower charges than interlinked peptides, we
also modified data acquisition control to select only higher
0
10
n
products behave similarly during sample preparation and MS
analysis, thus providing flexibility of using these isotope-coded
reagents in XL-MS studies.
To further explore the capability of d - and d -DMDSSO for
0
10
quantitative analysis, we cross-linked cytochrome C with d0-
and d -DMDSSO separately, carried out their in-gel digestion,
n
charged ions (i.e., 3+ and up) for MS analysis. This allows the
10
n
instrument to carry out data-dependent MS acquisition toward
and then mixed the resulting peptide digests in five chosen d /
0
n
potentially interlinked peptides. Importantly, the concurrent
usage of the isotope-labeled cross-linkers permits easy detection
of cross-linked peptides and increases the identification of
interlinked peptides overall. Taken together, our current
workflow has proven its effectiveness in identifying cross-linked
peptides.
d ratios (i.e., 5:1, 2:1, 1:1; 1:2, 1:5) prior to LC−MS analysis.
10
In order to determine the relative abundance ratios, we
manually obtained extracted ion chromatograms (XIC) for five
selected d - and d -labeled cross-linked peptide pairs for each
0
10
sample. As an example, Figure 6A−E illustrates the overlay of
XICs for a representative d - and d -DMDSSO interlinked
0
10
Quantitation of d /d Labeled Cross-Linked Peptides. In
0
10
peptide pair in five samples mixed with different ratios, and the
corresponding MS spectra are shown in Figure 6F−J. On the
basis of the calculated area under XICs, its relative abundance
addition to assisting MS detection and identification of cross-
linked peptides, we expect that isotope-coded cross-linkers can
be used to study protein structural changes by quantifying
relative abundances of nonlabeled and labeled cross-linked
peptides. In order to do this, protein cross-linking has to be
carried out using nonlabeled and labeled cross-linkers
separately assuming their cross-linking efficiencies are similar.
In our experiments, we have shown that cross-linking efficiency
(
d /d ) was determined as 4.79, 2.08, 0.99, 0.43, and 0.20,
0 10
respectively, which correlates well with the initial sample
mixing. In addition, the ratios obtained from peptide peak
intensity are similar to those obtained using XIC, indicating
that both approaches are sufficient for calculating relative
abundance of cross-linked peptides. As shown in Figure 6K, the
average ratios of the five selected cross-linked peptides for each
sample corroborate very well with initial sample mixing.
Collectively, these results have demonstrated the capability of
quantifying cross-linked peptides using isotope-coded
DMDSSO reagents.
of cytochrome C by d - or d -DMDSSO is very similar
0
10
(
Supplementary Figure 3 in the Supporting Information) and
equal mixing of the peptide digests of d - and d cross-linked
0
10
cytochrome C led to DMDSSO cross-linked peptide doublets
with relative ratios of 1 (Supplementary Figure 5A−C in the
Supporting Information). Since previous XL-MS studies often
cross-link proteins using a 1:1 mixture of nonlabeled and
labeled cross-linkers to generate isotopic pairs, we wanted to
compare whether equivalent results can be achieved using
different sample preparation approaches. Therefore, we first
CONCLUSIONS
■
We report here the development and characterization of new
DSSO derivatives, a pair of isotope-coded MS-cleavable cross-
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Analytical Chemistry
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linkers: d - and d -DMDSSO. DMDSSO cross-linked peptides
(11) Tang, X.; Munske, G. R.; Siems, W. F.; Bruce, J. E. Anal. Chem.
0
10
2
(
005, 77, 311−318.
preserve the same characteristic MS2 fragmentation patterns
distinctive to cross-link types as DSSO cross-linked peptides,
thus facilitating the detection and identification of cross-linked
peptides. In combination with MS3 sequencing, MS1 mass
mapping and isotopic profiling, the nature and identity of cross-
linked peptides can be determined readily and unambiguously.
This work further demonstrates the robustness of sulfoxide-
containing MS-cleavable cross-linkers in XL-MS studies and
provides a strong basis for us to further develop new DSSO
derivatives and fully define protein−protein interactions. In
addition, we have shown the flexibility in using d - and d -
12) Kao, A.; Chiu, C. L.; Vellucci, D.; Yang, Y.; Patel, V. R.; Guan,
S.; Randall, A.; Baldi, P.; Rychnovsky, S. D.; Huang, L. Mol. Cell.
Proteomics 2011, 10, M110 002212.
(
13) Back, J. W.; Notenboom, V.; de Koning, L. J.; Muijsers, A. O.;
Sixma, T. K.; de Koster, C. G.; de Jong, L. Anal. Chem. 2002, 74,
417−4422.
14) Muller, D. R.; Schindler, P.; Towbin, H.; Wirth, U.; Voshol, H.;
4
(
Hoving, S.; Steinmetz, M. O. Anal. Chem. 2001, 73, 1927−1934.
(15) Seebacher, J.; Mallick, P.; Zhang, N.; Eddes, J. S.; Aebersold, R.;
Gelb, M. H. J. Proteome Res. 2006, 5, 2270−2282.
(16) Krauth, F.; Ihling, C. H.; Ruttinger, H. H.; Sinz, A. Rapid
0
10
Commun. Mass Spectrom. 2009, 23, 2811−2818.
17) Petrotchenko, E. V.; Serpa, J. J.; Borchers, C. H. Mol. Cell.
Proteomics 2011, 10, M110 001420.
18) Rinner, O.; Seebacher, J.; Walzthoeni, T.; Mueller, L. N.; Beck,
M.; Schmidt, A.; Mueller, M.; Aebersold, R. Nat. Methods 2008, 5,
DMDSSO for quantitative analysis of cross-linked peptides,
thus establishing a solid foundation for our future studies
toward the understanding of structural dynamics of protein
complexes.
(
(
3
15−318.
ASSOCIATED CONTENT
Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
(19) Petrotchenko, E.; Borchers, C. BMC Bioinf. 2010, 11, 64.
(20) Yang, B.; Wu, Y. J.; Zhu, M.; Fan, S. B.; Lin, J.; Zhang, K.; Li, S.;
*
S
Chi, H.; Li, Y. X.; Chen, H. F.; Luo, S. K.; Ding, Y. H.; Wang, L. H.;
Hao, Z.; Xiu, L. Y.; Chen, S.; Ye, K.; He, S. M.; Dong, M. Q. Nat.
Methods 2012, 9, 904−906.
(
21) Singh, P.; Shaffer, S. A.; Scherl, A.; Holman, C.; Pfuetzner, R. A.;
AUTHOR INFORMATION
Corresponding Author
E-mail: lanhuang@uci.edu. Phone: (949) 824-8548. Fax:
949) 824-8540.
■
Larson Freeman, T. J.; Miller, S. I.; Hernandez, P.; Appel, R. D.;
Goodlett, D. R. Anal. Chem. 2008, 80, 8799−8806.
(22) Chu, F.; Baker, P. R.; Burlingame, A. L.; Chalkley, R. J. Mol. Cell.
Proteomics 2010, 9, 25−31.
*
(
(
23) Soderblom, E. J.; Goshe, M. B. Anal. Chem. 2006, 78, 8059−
068.
24) Lu, Y.; Tanasova, M.; Borhan, B.; Reid, G. E. Anal. Chem. 2008,
0, 9279−9287.
25) Leonard, N. M.; Brunckova, J. J. Org. Chem. 2011, 76, 9169−
174.
26) Lee, Y. J.; Lackner, L. L.; Nunnari, J. M.; Phinney, B. S. J.
Proteome Res. 2007, 6, 3908−3917.
Notes
8
(
8
(
9
(
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We wish to thank members of the Huang and Rychnovsky
Laboratory, especially Eric Novitsky, for their help during this
study. We would like to thank Prof. A. L. Burlingame, Drs.
Robert Chalkley, Shenheng Guan, and Peter Baker at UCSF for
using Protein Prospector. We would also like to thank Tonya
Second and Dr. Michael Senko at ThermoFisher Scientific for
their help in improving data acquisition. This work was
supported by National Institutes of Health Grants
RO1GM074830 and R21CA161807 to L.H., R01GM106003
to L.H. and S. R., and R01AI099190-01 to Haoping Liu.
REFERENCES
■
(
(
1) Wells, J. A.; McClendon, C. L. Nature 2007, 450, 1001−1009.
2) Guerrero, C.; Tagwerker, C.; Kaiser, P.; Huang, L. Mol. Cell.
Proteomics 2006, 5, 366−378.
3) Guerrero, C.; Milenkovic, T.; Przulj, N.; Kaiser, P.; Huang, L.
Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 13333−13338.
4) Fang, L.; Kaake, R. M.; Patel, V. R.; Yang, Y.; Baldi, P.; Huang, L.
Mol. Cell. Proteomics 2012, 11, 138−147.
5) Leitner, A.; Walzthoeni, T.; Kahraman, A.; Herzog, F.; Rinner,
O.; Beck, M.; Aebersold, R. Mol. Cell. Proteomics 2010, 9, 1634−1649.
6) Herzog, F.; Kahraman, A.; Boehringer, D.; Mak, R.; Bracher, A.;
(
(
(
(
Walzthoeni, T.; Leitner, A.; Beck, M.; Hartl, F. U.; Ban, N.;
Malmstrom, L.; Aebersold, R. Science 2012, 337, 1348−1352.
(
7) Lasker, K.; Forster, F.; Bohn, S.; Walzthoeni, T.; Villa, E.;
Unverdorben, P.; Beck, F.; Aebersold, R.; Sali, A.; Baumeister, W. Proc.
Natl. Acad. Sci. U.S.A. 2012, 109, 1380−1387.
(
8) Kao, A.; Randall, A.; Yang, Y.; Patel, V. R.; Kandur, W.; Guan, S.;
Rychnovsky, S. D.; Baldi, P.; Huang, L. Mol. Cell. Proteomics 2012, 11,
566−1577.
9) Kalisman, N.; Adams, C. M.; Levitt, M. Proc. Natl. Acad. Sci.
U.S.A. 2012, 109, 2884−2889.
10) Chu, F.; Mahrus, S.; Craik, C. S.; Burlingame, A. L. J. Am. Chem.
Soc. 2006, 128, 10362−10363.
1
(
(
2
106
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