CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY TO
MAP THREE-DIMENSIONAL PROTEIN STRUCTURES AND
PROTEIN–PROTEIN INTERACTIONS
Andrea Sinz*
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy,
University of Leipzig, D-04103 Leipzig, Germany
Received 29 September 2005; received (revised) 15 November 2005; accepted 26 November 2005
Published online 13 February 2006 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20082
Closely related to studying the function of a protein is the analysis
of its three-dimensional structure and the identification of
interaction sites with its binding partners. An alternative
approach to the high-resolution methods for three-dimensional
protein structure analysis, such as X-ray crystallography and
NMR spectroscopy, consists of covalently connecting two
functional groups of the protein(s) under investigation. The
location of the created cross-links imposes a distance constraint
on the location of the respective side chains and allows one to
draw conclusions on the three-dimensional structure of the
protein or a protein complex. Recently, chemical cross-linking of
proteins has been combined with a mass spectrometric analysis of
the created cross-linked products. This review article describes
the most popular cross-linking reagents for protein structure
analysis and gives an overview of the different available
strategies that employ chemical cross-linking and different mass
spectrometric techniques. The challenges for mass spectrometry
caused by the enormous complexity of the cross-linking reaction
mixtures are emphasized. The various approaches described in
the literature to facilitate the mass spectrometric detection of
cross-linked products as well as computer software for data
analyses are reviewed. # 2006 Wiley Periodicals, Inc., Mass
Spec Rev 25:663–682, 2006
Keywords: chemical cross-linking; bottom-up approach; topdown approach; MALDI-TOF mass spectrometry; ESI mass
spectrometry; FTICR mass spectrometry; protein 3D structure;
protein–protein interactions
I. INTRODUCTION
With recent progress in genome-sequencing projects, the number
of identified proteins has dramatically increased during the past
few years. The physiological function of many of the newly
discovered proteins, however, remains unclear. Closely related to
the study of the function of a protein is the analysis of its threedimensional structure and the identification of its interaction
partners. In those cases where high-resolution methods for
————
Contract grant sponsor: Saxon State Ministry of Higher Education,
Research and Culture; Contract grant sponsor: Deutsche Forschungsgemeinschaft (DFG project Si 867/7-1).
*Correspondence to: Dr. Andrea Sinz, Biotechnological-Biomedical
Center, Faculty of Chemistry and Mineralogy, University of Leipzig,
Linnéstrasse 3, D-04103 Leipzig, Germany.
E-mail: sinz@chemie.uni-leipzig.de
Mass Spectrometry Reviews, 2006, 25, 663– 682
# 2006 by Wiley Periodicals, Inc.
structural analysis are applicable, such as X-ray crystallography
and NMR spectroscopy, the solved three-dimensional structure
of a protein gives insights into stable interactions within a protein
complex. Theoretical modeling might reveal further interactions,
using the known three-dimensional structures as a starting point.
An alternative approach is to build up a set of structurally defined
interactions by covalently connecting pairs of functional groups
within a protein or a protein complex. The location of the created
cross-links imposes a distance constraint on the location of
the respective side chains and allows drawing conclusions on the
distance geometries of a protein or a protein complex structure
(Young et al., 2000; Back et al., 2003; Sinz, 2003).
Analysis of cross-linked peptides by mass spectrometry
makes use of several advantages associated with MS analysis:
(1) The mass of the protein or the protein complex under
investigation is theoretically unlimited because it is the
proteolytic peptides that are analyzed (in case a bottom-up
strategy is employed). (2) Analysis is generally fast and, in
favorable circumstances, requires only femtomole amounts of
total protein. (3) It is possible to gain insights into threedimensional structures of proteins in solution and flexible regions
are readily identified. (4) Membrane proteins and proteins that
exist as mixtures of different species (post-translational modifications, splice variants) are amenable to analysis. (5) The broad
range of specificities available for crosslinking reagents towards
certain functional groups, such as primary amines, sulfhydryls, or
carboxylic acids, and the wide range of distances that different
cross-linking reagents can bridge, offer the possibility to perform
a wide variety of experiments (Hermanson, 1996). When
selecting a specific reagent with a certain spacer length, one
should be aware that the average span of a cross-linker can be less
than the maximum calculated distance, according to stochastic
dynamics calculations (Green, Reisler, & Houk, 2001).
However, despite the straightforwardness of the crosslinking approach, the identification of the cross-linked products
can be quite cumbersome due to the complexity of the reaction
mixtures. Several strategies have been employed to enrich crosslinker-containing species by affinity chromatography or to
facilitate the identification of the cross-linked products; for
example by using isotope-labeled cross-linkers or proteins,
fluorogenic cross-linkers, or cleavable cross-linkers.
A number of reviews have been published recently that
either focus on chemical cross-linking reagents and application
protocols (Brunner, 1993; Kluger & Alagic, 2004; Melcher,
2004; Kodadek, Duroux-Richard, & Bonnafous, 2005) or on the
identification of protein–protein interactions, using chemical
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cross-linking combined with a mass spectrometric analysis of the
cross-linked products (Back et al., 2003; Sinz, 2003; Friedhoff,
2005; Trakselis, Alley, & Ishmael, 2005). The issue of using the
obtained information as a basis to create structural models of the
protein complexes has been addressed as well (Van Dijk,
Boelens, & Bonvin, 2005) and cross-linking combined with
mass spectrometry has been used to distinguish between the
crystallographic and physiological interface in dimeric proteins
(Petrotchenko et al., 2001).
In this review article, the most popular cross-linking
reagents for protein structure analysis are described and an
overview is given on the different available strategies that employ
chemical cross-linking and mass spectrometry. This article
highlights the challenges for mass spectrometry caused by the
enormous complexity of cross-linking reaction mixtures. Thus,
the various approaches described in the literature to facilitate
mass spectrometric detection of cross-linked products as well as
computer software for data analysis are reviewed. The present
review is not intended to give a comprehensive overview of the
dramatically increasing number of reports on the analysis of
protein structures, using well-established cross-linkers and MS
methods, but it aims to give insight into novel and promising
strategies. Application of those novel strategies to ‘real-life’
problems will show those proposed methods that will prevail.
II. CROSS-LINKING STRATEGIES
To conduct chemical cross-linking experiments of proteins, two
alternative strategies exist in principle, which is commonly
referred to as bottom-up and top-down approaches. In recently
developed innovative strategies, cross-linking reactions are
conducted in living cells by directly incorporating reactive
groups into the protein, using the cell’s own biosynthetic
machinery. In the following paragraph, the three strategies will
be described and compared to each other with respect to the
strengths and limitations of each strategy.
A. Bottom-Up Approach
In the bottom-up approach, the protein reaction mixture is
enzymatically digested after the cross-linking reaction, and mass
spectrometric identification of the cross-linked products is
performed, based on the resulting proteolytic peptides
(Fig. 1A). The bottom-up approach has been applied to map
protein interfaces, but it has also proven especially valuable to
determine low-resolution three-dimensional structures of proteins (Young et al., 2000; Sinz, 2003). The most important
prerequisite to successfully conduct crosslinking experiments is
a detailed description of the respective amino acid sequences of
the proteins under investigation. Full sequence coverage should
be envisioned to fully characterize the protein with respect to
possible amino acid variants, post-translational modifications, or
splice variants. When conducting cross-linking reactions, control
samples must be included, to which no cross-linker is added, to
exclude the formation of any non-specific aggregates. Moreover,
cross-linker concentrations, reaction times, and buffer pH must
be optimized to achieve a high yield of cross-linked product,
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while not disrupting the three-dimensional protein structures by
introducing too many cross-links per molecule.
After the cross-linking reaction, one-dimensional gel
electrophoresis (SDS–PAGE) and MALDI-TOF (matrixassisted laser desorption/ionization time-of-flight) MS analysis
of the reaction mixture can be used to check for the extent of
cross-linked product formation and to optimize the reaction
conditions. After the cross-linking reaction, there are several
ways to isolate the cross-linked proteins from the reaction
mixture. If SDS–PAGE of the cross-linking reaction mixture is
performed, the band of the cross-linked protein or the crosslinked protein complex is excised from the 1D-gel and subjected
to enzymatic in-gel digestion (Fig. 1A). Based on the staining
intensity of the respective gel bands, the amount of cross-linked
product formation is approximated. Alternatively, the crosslinked protein or protein complex is separated from the reaction
mixture by size-exclusion chromatography, and the digestion is
performed in the solution (Fig. 1A). We found that an in-solution
digestion of the cross-linked proteins is far more efficient than
in-gel digestion, where 80% of the protein is lost. The resulting
highly complex peptide mixtures generated from enzymatic
digestion contained unmodified peptides of the protein(s),
peptides modified by partially hydrolyzed cross-linker, and
intramolecular (inter- and intra-peptide) cross-linked products
between peptides originating from one protein as well as
intermolecular cross-linked products between peptides from
different proteins (Fig. 1A). Peptide mixtures that originated
from proteolytic digestion of cross-linking reaction mixtures
were analyzed by MALDI or ESI mass spectrometry. The crosslinked peptides were assigned in the mass spectra, using
customized software programs, such as the GPMAW software
(available at: http://welcome.to/gpmaw) (Peri, Steen, & Pandey,
2001). Based on signals in the mass spectra of cross-linking
mixtures, but not in those of control samples from noncross-linked proteins, cross-linked products were identified to
ultimately provide further information on the spatial
distances between functional groups of the protein(s) under
investigation.
One of the inherent problems of the bottom-up strategy is
that large peptides are commonly created from cross-linked
proteins during enzymatic proteolysis due to a high frequency of
missed cleavages. Missed cleavages occur because the most
commonly employed cross-linking reagents react with primary
amine groups at lysine residues and the N-termini of proteins, and
trypsin—the most commonly used proteolytic enzyme—will not
cleave C-terminal to a modified lysine residue. Another
limitation of the bottom-up approach is that cross-linked
products with low charge states are frequently created during
electrospray ionization due to a loss of positive charge after
modification of the e-amino groups of lysine residues; that
modification might cause large peptides not to be detected.
Moreover, the number of peptides with the same nominal mass
but different amino acid sequence, increases with the rising
number of amino acid residues in the peptide. Thus, mass
spectrometric techniques, which yield high resolution and high
mass accuracy data, and moreover allow fragmentation of large
peptides in MS/MS experiments, are a critically important
prerequisite for an unambiguous assignment of cross-linked
products.
CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES
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FIGURE 1. General analytical strategies for protein structure characterization by chemical cross-linking
and mass spectrometry. A: Bottom-up approach, and (B) top-down approach using FTICR-MS. Figure
adapted from Sinz (2005) with kind permission of Springer Science and Business Media.
B. Top-Down Approach
One of the most direct techniques to analyze cross-linked
products is the top-down approach, in which the cross-linked
proteins are analyzed intact rather than being digested before the
mass spectrometric analysis (McLafferty et al., 1999; Kelleher
et al., 1999) (Fig. 1B). Electrospray ionization-Fourier-transform
ion-cyclotron resonance (ESI-FTICR) mass spectrometry is the
method of choice for this kind of analysis. So far, the top-down
approach has been exclusively employed to determine lowresolution three-dimensional structures of proteins from intramolecular cross-linking experiments (Kruppa, Schoeninger, &
Young, 2003; Novak et al., 2003; 2005). The cross-linking
reaction mixture is presented to the FTICR mass spectrometer,
and the cross-linked product is isolated in the ICR cell before it is
interrogated with one of the various fragmentation techniques,
such as sustained off-resonance irradiation collision-induced
dissociation (SORI-CID), infrared multi-photon dissociation
(IRMPD), or electron capture dissociation (ECD) (Fig. 1B).
Instruments such as the novel commercially available hybrid
FTICR mass spectrometers additionally offer the possibility to
select and fragment ions prior to the ICR cell. Determination of
the accurate mass of the intact cross-linked product provides
hints on the number of incorporated cross-linker molecules as
well as on the number of modifications caused by partially
hydrolyzed cross-linkers. The top-down approach presents some
advantages over the bottom-up approach in that it eliminates the
need to separate the reacted protein from the cross-linking
reaction mixture before the mass spectrometric analysis, because
this separation is accomplished by a ‘gas-phase purification’ in
the mass spectrometer. After fragmentation of the cross-linked
protein in the FTICR mass spectrometer, assignment of the crosslinked products is performed manually or by customized
software programs (MS2PRO, available at: http://roswell.ca.sandia.gov/mmyoung/). Top-down approaches have been
successfully employed to assign intramolecular crosslinked
products of bovine rhodopsin (Novak et al., 2005) as well as
ubiquitin (Kruppa, Schoeninger, & Young, 2003; Novak et al.,
2003). ECD seems to be especially favorable in conjunction with
FTICR-MS because it allows a comprehensive fragmentation of
large peptides, while post-translational modifications are kept
intact. One limitation of the top-down approach is that analyses of
large protein assemblies are difficult to perform. In the case of
characterizing bovine rhodopsin (Novak et al., 2005), the protein
was proteolyzed into large peptide fragments, using cyanogen
bromide, which cleaves at the C-terminal site of methionine
residues, before ESI-FTICR-MS/MS experiments were conducted in a top-down fashion. That combination of bottom-up
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and top-down analysis will most likely become the strategy with
the greatest potential for a rapid and efficient analysis of a wide
variety of cross-linking reaction mixtures.
C. Chemical Cross-Linking in Living Systems
Very recently, several highly appealing approaches have been
developed, which permit cross-linking of interacting proteins in
their natural environment, and thus, give insight into the way
cellular processes are organized.
In one strategy reported, live cells are treated with
formaldehyde, which rapidly permeates the cell membrane and
generates cross-links between interacting proteins in the
cell. Proteins that are cross-linked to a myc-tagged protein of
interest are co-purified by immunoaffinity chromatography,
the cross-linked complexes are subsequently dissociated, the
bound proteins are separated by one-dimensional gel electrophoresis and identified by tandem MS (Vasilescu, Guo, & Kast,
2004).
Another intriguing strategy is based on the incorporation of a
unique chemical into a protein of interest, using the cell’s own
biosynthetic machinery (Prescher & Bertozzi, 2005), followed by
a chemical reaction with a small-molecule probe. So far, only a
handful of chemical motifs are known to possess the requisite
qualities of biocompatibility and selective reactivity to function
as chemical reporters in living cells. Such chemical motifs
include the tetra-Cys motif that reacts selectively with biarsenicals or azides that react with phosphines in a Staudinger ligation
or in a copper-catalyzed ‘click’ chemistry type reaction with
alkynes (Prescher & Bertozzi, 2005). In another interesting
study, three new photoactivatable amino acids that contain a
diazirine moiety were designed and termed ‘‘photo-methionine’’
and ‘‘photo-leucine,’’ and ‘‘photo-isoleucine’’ (Suchanek,
Radzikowska, & Thiele, 2005) (Fig. 2). The structural similarity
to the natural amino acids methionine, leucine, and isoleucine
allows the artificial amino acids to escape the stringent identity
control mechanisms of a cell during protein synthesis and to be
incorporated into proteins by the cell’s translation machinery.
Activation by UV light induces covalent cross-linking (see
paragraph ‘photoreactive cross-linkers’). The preference for
methionine, leucine, and isoleucine implies that transmembrane
domains as well as hydrophobic contact areas between proteins
are preferentially cross-linked. In this preference, the method is
complementary to the most commonly employed amine-reactive
chemical cross-linkers that target lysine residues and the
N-termini of proteins. A mass spectrometric identification of
cross-linked products created in a living cell, however, has not
been described so far and is likely to be challenging.
III. FUNCTIONAL GROUPS OF
CROSS-LINKING REAGENTS
A. Reactivities
Chemical cross-linking reactions form covalent bonds between
different molecules (intermolecular) or within parts of a
molecule (intramolecular). The hundreds of reagents described
in the literature (Wong, 1991; Hermanson, 1996) or offered
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FIGURE 2. Chemical structures of the three new amino acids
‘photo-Ile,’ ‘photo-Leu,’ and ‘photo-Met’(left hand side) in comparison
to the natural amino acids Ile, Leu, and Met (right-hand side).
commercially are based on a small number of organic chemical
reactions, thus reducing the number of functionalities in the
protein that can react with the cross-linker. In the following
paragraph, the most widely used classes of cross-linking reagents
are described with respect to their specific strengths and
limitations.
1. Amine-Reactive Cross-Linkers
a. N-hydroxysuccinimide esters. N-hydroxysuccinimide
(NHS) esters are probably the most widely applied principle
to create reactive acylating reagents. Thirty years ago, NHS
esters were introduced as homobifunctional, highly aminereactive, cross-linking reagents (Bragg & Hou, 1975; Lomant &
Fairbanks, 1976). Because many NHS esters are insoluble in
aqueous buffers, most protocols involve dissolving the NHS
ester in an organic solvent at high concentration and diluting that
stock solution in the reaction medium. Alternatively, sulfo-NHS
esters—the water-soluble analogs—are used. NHS esters react
with nucleophiles to release the NHS or sulfo-NHS group and to
create stable amide and imide bonds with primary or secondary
amines, such as free N-terminus and e-amino groups in lysine
side chains of proteins (Scheme 1A). NHS esters exhibit halflifes in the order of hours under physiological pH conditions (pH
7.0–7.5) with hydrolysis and amine reactivity increasing when
the pH is raised (Cuatrecaseas, 1972; Lomant & Fairbanks,
1976; Staros, 1988; Hermanson, 1996). By varying the molar
ratio of cross-linking reagent and protein the level of
modification is adapted to the requirements of the individual
application.
According to the older literature (Hermanson, 1996), the
reaction of NHS esters with sulfhydryl groups (in cysteines) or
CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES
hydroxyl groups (e.g., in serine or threonine) does not yield stable
products because the thioester or ester products hydrolyze
rapidly in aqueous solution. Imidazole nitrogens of the histidine
ring might also be acylated by NHS esters, but the reaction
products are also rapidly hydrolyzed (Cuatrecaseas & Parikh,
1972). Based on our own experience with NHS esters, however,
we frequently observed cross-linked products of serine hydroxyl
groups in MS analysis, so the esters were apparently sufficiently
stable in aqueous solution. This observation was confirmed by a
carefully conducted study, using the homobifunctional, aminereactive, and cleavable cross-linker DTSSP (3,30 -dithiobis[sulfosuccinimidyl propionate]) (Swaim, Smith, & Smith, 2004).
Reaction products of several model peptides with DTSSP were
analyzed by ESI-QqTOF mass spectrometry and reaction
products were confirmed by tandem MS experiments. The NHS
ester DTSSP was found to react unexpectedly with contaminant
ammonium ions in the buffer solution and with serine and
tyrosine residues in addition to the desired reactions with lysine
residues and the N-terminus (Swaim, Smith, & Smith, 2004).
Another study describes the formation of stable products when
NHS esters reacted with primary amines and tyrosine OH groups
(Leavell et al., 2004). Under acidic conditions (pH 6.0), the NHS
esters were found to react preferentially with the N-terminus and
tyrosine hydroxyl groups; however, under alkaline conditions
(pH 8.4) they were found to react preferentially with the Nterminus and lysine amine groups. These findings underline the
urgent need to conduct further research on the reactivities of NHS
esters, and cross-linking reagents in general; those studies have
unfortunately been largely neglected so far.
b. Imidoesters. The imidate functional group is one of the
most specific acylating groups to modify primary amines, and
cross-linking reagents that contain imidoesters at both ends
are among the oldest reagents used for protein conjugation
(Scheme 1B) (Hartmann & Wold, 1966). Unlike most other
cross-linking reagents, imidoesters possess minimal cross
reactivity towards other nucleophiles. The N-terminus of the
protein as well as the e-amino groups in lysine side chains react
best at a pH between 8 and 9. Imidoesters are highly watersoluble, but undergo continuous degradation due to hydrolysis,
which reduces the half-life of the imidate group to less than
30 min (Hunter & Ludwig, 1962; Browne & Kent, 1975). The
reaction product of the imidoester, an amidine, is protonated and
carries a positive charge at physiological pH (Liu, Fairbanks, &
Palek, 1977; Kiehm & Ji, 1977; Wilbur, 1992). Retaining the
positive charge of the lysine group presents one of the major
advantages of imidoesters because one of the greatest dangers to
distort the three-dimensional structure of proteins arises from
removing the positive charge at the lysine residues when amino
groups are targeted in cross-linking reactions. From our
experience, using imidoesters for cross-linking of proteins at
physiological pH (Dihazi & Sinz, 2003), however, we found only
a modest cross-linking efficiency compared to NHS esters;
therefore, we consider NHS esters to be superior when amine
groups are targeted.
c. Carbodiimides. Carbodiimides are so-called ‘zero-length’
cross-linking reagents because they do not introduce a spacer
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chain into the protein. They are used to mediate amide bond
formation between spatially close (<3 Å) groups; for example, a
carboxylate and an amine group or a phosphate and an amine
group (Hoare & Koshland, 1966; Chu, Kramer, & Orgel, 1986;
Ghosh et al., 1990). A second reagent such as sulfo-Nhydroxysuccinimide could be added to modify the reaction
(Scheme 1C). Carbodiimide-mediated amide formation occurs
effectively between pH 4.5 and 7.5.
2. Sulfhydryl-Reactive Cross-Linkers
The problem while targeting SH groups of cysteines is their
possible involvement in disulfide-bond formation. Reduction of
disulfide bonds to create free SH groups implies the danger of
distorting the three-dimensional protein structure.
a. Maleimides. Maleic acid imides (or maleimides) are a
widely used reactive group in many heterobifunctional crosslinking reagents. The thiolate anion is added to the maleimide,
according to Scheme 1D. Maleimide reactions are sulfhydrylspecific in the pH range between 6.5 and 7.5 (Smyth, Konigsberg,
& Blumenfeld, 1964; Gorin, Matic, & Doughty, 1966; Heitz,
Anderson, & Anderson, 1968; Partis et al., 1983), and at pH 7 the
reaction with maleimides proceeds ca. 1,000-times faster with
sulfhydryls than with amines. At more basic pH values, however,
reactions with amines might also take place (Brewer & Riehm,
1967). Hydrolysis of the maleimide group might occur especially
at higher pH to create an open maleamic form, which is nonreactive towards sulfhydryl groups.
3. Photoreactive Cross-Linkers
Photoreactive cross-linkers are induced to react with target
molecules by exposure to UV light. The ideal photoreactive agent
should be of high reactivity, capable of indiscriminately inserting
into any type of residue, stable in the dark, and highly susceptible
to light of a wavelength that does not cause any photolytic
damage to the biological sample. Moreover, the reaction with
proteins should lead to stable and unique products to enable their
isolation, purification, and subsequent mass spectrometric
analysis. By far, the largest number of photoreactive reagents is
based on nitrene or carbene chemistry with the photolabile
precursors being azides, diazirines, diazo compounds, and
benzophenones. Most of the photoreactive cross-linkers are
heterobifunctional reagents, which additionally possess an
amine- or sulfhydryl-reactive group (Pierce Perbio, 2003/
2004).
a. Aryl azides. Phenyl- and nitro-substituted phenyl azides
have played a leading role in many areas of photochemical
labeling, and currently they constitute the most commonly
employed class of photoreactive cross-linkers. Upon photolysis,
phenyl azide groups form short-lived nitrenes that can insert nonspecifically into chemical bonds of target molecules, including
addition reactions at double bonds and insertion reactions into
active hydrogen bonds at C–H and N–H sites (Scheme 1E)
(Gilchrist & Rees, 1969). The major disadvantages of aryl azides,
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SCHEME 1. Reaction schemes of the most commonly used reagents for cross-linking of proteins. A: NHS esters (amine-reactive), (B) imidates
(amine-reactive), (C) ‘zero-length’ cross-linker EDC in combination with sulfo-NHS (amine/carboxylic acid-reactive), (D) maleimides (sulfhydrylreactive), (E) aryl azides (photoreactive), (F) diazirines (photoreactive), (G) benzophenones (photoreactive), and (H) tris(2,20 -bipyridyl)ruthenium (II)
dication (photoreactive).
however, are that they are activated by short-wavelength UV
irradiation (<280 nm) and that the intermediately created nitrene
may react non-uniformly.
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b. Diazirines. Diazirines are remarkably stable to a variety of
chemical conditions, and are efficiently photolyzed at wavelengths of ca. 360 nm to generate a highly reactive carbene
CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES
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SCHEME 1. (Continued)
that can insert into a heteroatom-H or C–H bond (reaction
Scheme 1F). Unfortunately, photolysis of diazirines might lead to
diazo isomers, which present strongly alkylating species that are
responsible for undesired reactions in the dark (Brunner, 1993).
c. Benzophenones. A completely different photochemistry
compared to aryl azides or diazirines is exhibited by benzophenones (Dorman & Prestwich, 1994; Egnaczyk et al., 2001; Junge
et al., 2004), which create a biradical upon irradiation.
Subsequently, the oxygen radical abstracts a hydrogen radical
from a bond of the reaction partner (Scheme 1G). The alkyl
radicals created react by forming a new C–C bond between the
photophor and the receptor protein. In contrast to diazirine
compounds, activation of benzophenones does not proceed
according to a photo-dissociative mechanism and is, therefore,
reversible.
d. Photo-induced cross-linking of unmodified proteins
(PICUP). Recently, oxidative crosslinking techniques have
been developed that are mediated by high-valent metal–chelate
complexes. The main advantage of that type of reaction is that
they are extremely rapid and do not require a chemical
modification of the protein (Kodadek, Duroux-Richard, &
Bonnafous, 2005). The high-valent metal complexes employed
are derived from stable, lower-valent species, such as His6-Ni2þ,
Gly-Gly-His-Ni2þ, or tris(2,20 -bipyridyl)ruthenium (II) dication
([Ru(bipy)3]2þ) (Kodadek, Duroux-Richard, & Bonnafous,
2005). Light irradiation of the water-soluble ruthenium complex
[Ru(bipy)3]2þ in the presence of single-electron acceptors
mediates fast and high-yield cross-linked product formation of
proteins. That process has been termed ‘Photo-induced CrossLinking of Unmodified Proteins’ (PICUP) (Gerardi, Barnett, &
Lewis, 1999; Bitan & Teplow, 2004). When photo-excitation is
performed in the presence of an electron acceptor, Ru(II) is
oxidized to Ru(III). Ru(III) is a strong one-electron oxidizer that
abstracts an electron from a nearby protein molecule to produce a
protein radical and recycling back to Ru(II) (Scheme 1H).
Radical formation might occur at any site along the polypeptide
chain; however, the radical will form preferentially at side chains
that offer stabilization through aromatic or neighboring group
effects, such as histidine, tyrosine, or cysteine residues. Once the
protein radical is created, it can attack a neighboring protein to
create an intermolecular cross-link (Scheme 1H). One of the
major advantages of the PICUP method is that the reaction
requires very short time period (less than 1 sec) and that it occurs
across a wide pH range. The cross-linking efficiency is usually
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high and the ability of the reagent to function as a ‘zero-length’
cross-linker makes it an attractive tool to take ‘snapshots’ of
dynamic protein associations. The PICUP strategy has been
employed to study the structure, kinetics, and thermodynamics of
the formation of metastable protein oligomers. Those soluble
oligomers are precursors for amyloid fibrils that are the primary
toxic effectors responsible for the disease process in amyloidoses, such as Alzheimer’s disease (Bitan & Teplow, 2004).
B. Cross-Linker Design
1. Homobifunctional Cross-Linkers
Homobifunctional cross-linking reagents contain identical functional groups at both reactive sites, which are connected with a
carbon-chain spacer that bridges a defined distance (Fig. 3A) and
thus, allows identical functional groups of proteins (e.g., amine or
sulfhydryl groups) to be cross-linked. The variety of homobifunctional cross-linking reagents has increased dramatically
during the past 25 years, and today, a wide variety of reagents are
commercially available that possess different spacer lengths
and reactivities (product catalogs available, e.g., at http://www.
piercenet.com/ and http://www.trc-canada.com/).
The main disadvantage of homobifunctional reagents is their
susceptibility to create a wide range of poorly defined products
(Avrameas, 1969). The cross-linking reagent reacts initially with
a protein molecule to form an intermediate, which could react
with a second protein molecule to create a high-molecular weight
aggregate, or which alternatively, could react intramolecularly
with a neighboring functional group on the same polypeptide
chain. Maintaining a protein concentration in the mM range
during the reaction is generally desirable in that it reduces
unwanted intermolecular cross-linking between proteins. To
check for high-molecular weight aggregates due to intermolecular cross-linking, one-dimensional gel electrophoresis as well
as a rapid mass spectrometric screening, for example, by
MALDI-TOF-MS should be performed to establish the optimum
cross-linking reaction conditions for the different cross-linking
reagents. Special caution has to be applied not to disturb the
three-dimensional structure of the proteins by excessive crosslinking, but on the other hand, sufficient amounts of cross-linking
products have to be created to allow for a subsequent mass
spectrometric detection.
Especially single-step reaction procedures, using homobifunctional cross-linking reagents, in which all reagents are
added at the same time to the reaction mixture, pose the greatest
potential to form a multitude of different cross-linked products.
To overcome that limitation, two-step protocols have been
developed, in which one of the proteins is first reacted with the
cross-linker to form an ‘activated’ protein. After the reaction,
excess reagent is removed and the activated protein is mixed with
the second protein for cross-linking (Hermanson, 1996).
2. Heterobifunctional Cross-Linkers
Heterobifunctional cross-linking reagents contain two different
reactive groups that target different functional groups on
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FIGURE 3. Different types of cross-linking reagents. A: Homobifunctional cross-linker, (B) heterobifunctional cross-linker, (C) trifunctional
cross-linker, and (D) heterobifunctional, cleavable cross-linker.
proteins; for example, an amine and a sulfhydryl group
(Fig. 3B). Those cross-linking reagents are used to cross-link
proteins favorably in two- or three-step protocols to minimize the
degree of high-molecular weight aggregate formation. For
example, an NHS ester/maleimide heterobifunctional crosslinker can be applied for reaction with a protein amine group at its
NHS ester function, whereas the maleimide group does not react
due to its higher stability in aqueous solution. After a purification
step, the maleimide function of the cross-linker can react with a
protein sulfhydryl group. Heterobifunctional cross-linkers that
contain one photoreactive group offer additional advantages,
because the photoreactive group is stable until it is exposed to
high intensity UV light.
3. Zero-Length Cross-Linkers
The smallest reagent systems available for chemical crosslinking are zero-length cross-linkers. Those compounds mediate
cross-linking between two proteins by creating a bond without an
intervening linker. Carbodiimides are probably the most widely
used type of zero-length cross-linkers, which are applied to
mediate amide bond formation between a carboxylate and an
amine group (or a phosphoramidate bond formation between a
phosphate and an amine) (Hoare & Koshland, 1966; Ghosh et al.,
CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES
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1990); EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)
is the most popular representative (Hermanson, 1996). EDC is
mostly applied in combination with sulfo-NHS (Staros, Wright,
& Swingle, 1986). The purpose of adding sulfo-NHS to EDC is to
increase the stability of the active intermediate, which ultimately
reacts with the amine group according to Scheme 1C. EDC/sulfoNHS-coupled reactions are highly efficient and usually increase
the yield of cross-linked product formation compared to EDC
alone (Staros, Wright, & Swingle, 1986). PICUP reagents must
also be considered as zero-length cross-linkers.
4. Trifunctional Cross-Linkers
The trifunctional cross-linker approach incorporates elements of
the heterobifunctional cross-linker concept with the additional
third functional group being able to specifically link to a third
protein or being used for affinity purification of cross-linker
containing species in case a biotin moiety is incorporated
(Trester-Zedlitz et al., 2003; Fujii et al., 2004) (Fig. 3C). In an
excellent study conducted by Trester-Zedlitz et al., five
trifunctional cross-linking reagents were synthesized, including
two or more of the following groups: an amine-reactive NHS
ester, a sulfhydryl-reactive maleimide, a photochemically
reactive benzophenone, an isotope tag, a biotin handle, and/or a
baselabile ester cleavage site. The incorporation of isotope labels
into a biotinylated, trifunctional cross-linker is likely to become
one of the most promising strategies to design novel cross-linking
reagents. One of the few commercially available biotinylated
cross-linking reagents is sulfo-SBED (sulfosuccinimidyl-2-[6(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,30 dithiopropionate) (Scheme 2). That trifunctional cross-linker
possesses one amine-reactive and one photoreactive site, is
cleavable by reducing agents, and additionally allows an
affinity-based enrichment of cross-linker-containing species.
IV. MASS SPECTROMETRIC ANALYSIS
OF CROSS-LINKED PRODUCTS
The main challenges to identify cross-linked products by mass
spectrometry arise from the high complexity of the reaction
mixtures. The soft-ionization techniques MALDI (Karas &
Hillenkamp, 1988) and ESI (electrospray ionization) (Fenn et al.,
1989) are the predominately employed methods to analyze crosslinking mixtures of proteins.
A. Bottom-Up Analysis by MALDI-MS
The mechanisms of ion formation in MALDI are a subject of
continuing research (Zenobi & Knochenmuss, 1999; Menzel
et al., 2001; Dreisewerd et al., 2001; Karas & Krüger, 2003).
MALDI generated a great demand for a mass analyzer ideally
suited to be used in conjunction with a pulsed ion source, such as
the time-of-flight (TOF) analyzer. The performance of TOF
instruments has increased tremendously during the past few
years. Two true tandem TOF instruments have become
commercially available (Schnaible et al., 2002; Yergey et al.,
2002) and are likely to be beneficial to analyze cross-linked
SCHEME 2. Chemical structure of the trifunctional cross-linker sulfo-
SBED.
products. By conducting MS/MS experiments of cross-linked
peptides, sequence information of the cross-linked peptides
and information on the sites of cross-linking both become
available. MALDI-TOF-MS has been applied in numerous
studies to analyze cross-linking reaction mixtures; for example
as described by Bennett et al. (2000), Rappsilber et al. (2000),
Young et al. (2000), Cai, Itoh, & Khorana (2001), Egnaczyk et al.
(2001), Itoh, Cai, & Khorana (2001), Müller et al. (2001);
Pearson, Pannell, & Fales (2002), Sinz & Wang (2001); Back
et al. (2002a,b), D’Ambrosio et al. (2003), Trester-Zedlitz et al.
(2003), Wine et al. (2002), Chang, Kuchar, & Hausinger (2004),
Giron-Monzon et al. (2004), and Onisko et al. (2005). In one
report, MALDI-quadrupole ion trap (QIT) mass spectrometry
has been employed to identify cross-linked products (Peterson,
Young, & Takemoto, 2004).
B. Bottom-Up Analysis by ESI-MS (LC/MS)
In ESI, liquids are sprayed in the presence of a strong electric
field, forming small, highly charged droplets. ESI requires a
sample that is devoid on non-volatile salts and detergents to
obtain the highest sensitivity as well as careful optimization of
electrospray conditions for the specific compound under
investigation. Miniaturization of the electrospray technique
(nano-electrospray), by applying narrower spray capillaries,
results in smaller droplets, reduced flow rates, and improved
sensitivity (Wilm & Mann, 1994, 1996). The peptide mixture is
usually introduced into the mass spectrometer by a separation
technique such as liquid chromatography (LC) or capillary
electrophoresis (CE). Complex peptide mixtures are mostly
separated by reversed-phase high performance liquid chromato671
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graphy (RP-HPLC). ESI-MS/MS is the method of choice to
obtain further information on the amino acid sequence of crosslinked peptides as well as on the cross-linked amino acids. MS
experiments using ESI-QIT, ESI-QqTOF, or ESI-TOF instruments are frequently used for a detailed analysis of cross-linked
products (Chen, Chen, & Anderson, 1999; Back et al., 2001,
2002a,b; Egnaczyk et al., 2001; Müller et al., 2001; Sinz & Wang,
2001; Pearson, Pannell, & Fales, 2002; Lanman et al., 2003;
Taverner et al., 2002; Trester-Zedlitz et al., 2003; Huang, Kim, &
Dass, 2004; Swaim, Smith, & Smith, 2004; Füzesi et al., 2005;
Onisko et al., 2005; Silva et al., 2005).
MS/MS experiments, using CID, IRMPD, and ECD. In ECD,
reduced radical cations [M þ nH](n-1) þ . are generated upon
capture of electrons, which dissociate by fast and facile
fragmentation of the N–Ca bond of the peptide chain, producing
mainly c and z. type fragment ions (Zubarev, Kelleher, &
McLafferty, 1998).
When analyzing intra-molecular cross-linked products of
bovine rhodopsin, only ECD revealed full palmitoylation of
adjacent cysteines and cross-linking of a lysine residue to two
other lysines and one cysteine residue based on the presence of a
number of crucial c- and z.-type ions (Fig. 5).
C. Bottom-Up and Top-Down
Analysis by ESI-FTICR-MS
V. IDENTIFICATION OF
CROSS-LINKED PRODUCTS
For a confident assignment of cross-linker containing species, the
application of high resolution and high mass accuracy methods,
such as FTICR mass spectrometry, is a valuable prerequisite
(Comisarov & Marshall, 1974; Marshall, 2000). With its ultrahigh resolution and mass accuracy FTICR-MS offers the
possibility of unambiguously identifying cross-linked species
based only on accurate mass measurements (Carlsohn et al.,
2004; Schulz et al., 2004). With the newest Q-TOF instruments,
however, which offer mass accuracies that begin to approach
those of FTICR mass spectrometers, it might be feasible to
analyze cross-linked products with similar mass accuracy.
In FTICR mass spectrometers, precursor-ion selection is
accomplished by storing the ions of interest, whereas all others
are ejected by means of a suitably tailored excitation pulse; for
example, using the SWIFT technique (Guan & Marshall, 1996).
MS/MS experiments are performed, using SORI-CID (Gauthier,
Trautman, & Jacobson, 1991), IRMPD (Little et al., 1994), or
ECD (Zubarev, Kelleher, & McLafferty, 1998; Zubarev, 2003).
Alternatively, with FTICR mass spectrometers that possess a
quadrupole or a linear ion trap in front of the ICR cell it is possible
to conduct MS/MS experiments prior to the ICR cell (Gershon,
2003). ESI-FTICR mass spectrometry has been coupled on-line
with capillary and nano-liquid chromatography for highthroughput peptide identification with high sensitivity (Shen
et al., 2001; Ihling et al., 2003). In our group, we have been using
nano-HPLC/nano-ESI-FTICR-MS to analyze cross-linking mixtures created from intra-molecular cross-linking of proteins
(Dihazi & Sinz, 2003) and from intermolecular cross-linking of
protein/peptide complexes, using a bottom-up approach (Schulz
et al., 2004; Kalkhof et al., 2005; Schmidt et al., 2005; Sinz,
Kalkhof, & Ihling, 2005). Using ambiguous distance restraints
derived from the chemical cross-linking data in combination with
recently developed computational methods of conjoined rigid
body/torsion angle-simulated annealing, we were able to
generate low-resolution three-dimensional structure models of
the calmodulin–melittin complex (Fig. 4), for which no highresolution structure exists to date (Schulz et al., 2004).
ESI-FTICR mass spectrometry is the method of choice for
top-down analyses (Fig. 1B) and has so far been successfully
employed to assign cross-linked products from intra-molecularly
cross-linked proteins, such as rhodopsin (Novak et al., 2005) or
ubiquitin. In the case of bovine rhodopsin, cyanogen bromide
fragments of the cross-linked protein were subjected to FTICR-
As mentioned above, mass spectrometric identification of crosslinked products can be hampered by the inherent complexity of
the cross-linking reaction mixtures. In Figure 6, the ESI-FTICR
mass spectrum of a cross-linking reaction mixture is shown, in
which the N-terminal domains (LN-domains) of laminin b1 and
laminin g1 (possessing molecular weights of ca. 59 and 55 kDa)
have been cross-linked using the homobifunctional, aminereactive cross-linker BS3. The signals of two peptides that have
been modified by a partially hydrolyzed cross-linker are presented as insets, thus, illustrating the low signal intensities of
cross-linker-containing species and the complexity of the mass
spectra. Searching for cross-linked peptides in a digestion
mixture, when a bottom-up analysis is applied, is comparable to
looking for the infamous ‘needle in the haystack.’ To locate the
‘needles’ (cross-linked products), a number of strategies have
been developed that aim to facilitate the identification of crosslinked products by introducing discriminating properties or by
enriching cross-linker-containing species, using specific tags.
The employed strategies utilize:
672
isotope-labeled cross-linkers or proteins,
cross-linkers with affinity tags,
fluorogenic cross-linkers, and
chemically or MS/MS cleavable cross-linkers.
A. Isotope-Labeling
1. Isotope-Labeled Cross-Linkers
Application of 1:1 (w/w) mixtures of stable isotope-labeled
cross-linking reagents allows low-abundance cross-linked peptides to be easily detected by their distinctive isotopic patterns
after enzymatic digestion (Müller et al., 2001; Pearson, Pannell,
& Fales, 2002). We employed the homobifunctional, aminereactive NHS esters BS3 (bis[sulfosuccinimidyl]suberate) and
BS2G (bis[sulfosuccinimidyl]glutarate) (Table 1) to map the
complex between calmodulin and a peptide derived from the
C-terminus of adenylyl cyclase 8 (Schmidt et al., 2005). BS3 and
BS2G were employed to conduct cross-linking reactions as 1:1
(w/w) mixtures with their deuterated derivatives (d0/d4) (Table 1).
Thus, an additional criterion for the identification of cross-linked
CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES
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FIGURE 4. Parallel (A) and anti-parallel (B) modes of binding of melittin in the calmodulin–melittin
complex calculated from ambiguous distance restraints derived from the cross-linking data using conjoined
rigid body/torsion angle-simulated annealing. The amino acid side chains of clamodulin and melittin that are
involved in cross-linking are shown for both orientations A and B. Each orientation is represented by two
views: One with the helix axis of melittin parallel (left hand side panels) and one with the helix axis of
melittin perpendicular (right hand side panels) to the plane of the paper. Reprinted from Schulz et al. (2004)
with permission, copyright American Chemical Society. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
products is introduced, because every species that contained one
cross-linker molecule exhibited a doublet with a mass difference
of 4.025 amu in the deconvoluted ESI-FTICR mass spectra.
The deconvoluted ESI-FTICR mass spectrum, from which
an intra-molecular cross-linked product of calmodulin was
identified, is presented in Figure 7.
Another promising strategy using 18O-labeling of the
homobifunctional amine-reactive cross-linker BS3 (Table 1)
has been reported (Collins et al., 2003). A bis-18O-labeled crosslinker was synthesized, so that proteins might be cross-linked by
a defined mixture of unlabeled and 18O-labeled reagent to
produce two sets of signals for crosslinked products that are
separated by 4 amu. Peptides that are modified by partially
hydrolyzed cross-linker are easily resolved by executing a
simultaneous experiment that used unlabeled BS3 in the presence
of H218O, resulting in a 2-amu mass shift for singly modified
peptides. Reactions were monitored by MALDI-TOF-MS and
ESI-TOF-MS analysis. Compared to deuterated cross-linkers,
that strategy possessed the advantage that 18O-labeled crosslinkers do not show any isotope effects in LC/MS analysis,
whereas deuterated cross-linkers might exhibit slightly different
retention times from their nondeuterated counterparts.
2. Isotope-Labeled Proteins
The first report of the use of isotope labeling of proteins for a
facilitated identification of cross-linked products involved a
rather complicated labeling procedure (Chen, Chen, & Anderson,
1999). All primary amino groups in the protein were reductively
methylated in a first reaction step, and after enzymatic hydrolysis,
the newly formed N-termini of the peptides were derivatized with
a 1:1 (w/w) mixture of 2,4-dinitrofluorobenzene-d0/d3. Only
cross-linked peptides reacted twice and could be discriminated in
the mass spectra by their 1:2:1 isotope pattern from the 1:1
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FIGURE 5. ECD-FTICR mass spectrum of an intramolecular cross-linked product of rhodopsin, using the
homobifunctional, amine-reactive NHS ester disuccinimidyl suberate [DSS]—A BS3 analog that lacks the
sulfo group. ECD was performed on the [M þ DSS þ 6H]6þ ion at m/z 1140.638, composed of peptides a
(upper amino acid sequence) and b2 (lower amino acid sequence) (molecular weight of cross-linked product
7977.421 Da). The spacing from c4b to c5b reveals full palmitoylation of the adjacent cysteine residues
Cys-322 and C-232. The analysis of this spectrum also reveals cross-linking of Lys-67 8and not Lys-66) to
Lys-325 and Lys-339. Reprinted from Novak et al. (2005) with permission, copyright 2005 American
Chemical Society.
pattern of the singly labeled non-cross-linked peptides using the
properties of the chromophore for chromatographic separation.
Another strategy to visualize intermolecular cross-linked peptides, using isotope-labeled proteins, was termed the ‘mixed
isotope cross-linking’ (MIX) strategy (Taverner et al., 2002).
That approach involved mixing 1:1 (w/w) 15N-labeled and
unlabeled (14N) protein to form a mixture of isotope-labeled and
non-labeled protein and was exemplified by using homodimers of
cytokine interleukin 6 (14N, 14N/15N, and 15N), which were
chemically cross-linked. Intermolecular cross-linked peptides
were identified by their distinctive triplets or quadruplets in their
ESI mass spectra. In contrast, intramolecularly cross-linked or
non-cross-linked peptides appeared as doublets in the mass
spectra, thus offering the possibility to discriminate between
inter- and intramolecular cross-linked species.
3.
18
O-Labeling During Proteolytic Digestion
Incorporation of 18O from isotopically enriched water into the Ctermini of proteolytic peptides also presents a valuable means to
discriminate cross-linked products. The mass shifts rely on the
complete incorporation of two 18O atoms during proteolytic
hydrolysis with trypsin for each C-terminus of a lysine-or
arginine-containing peptide. Acid- and base-catalyzed backexchange with concomitant loss of the isotope label can occur at
extreme pH values, but under the mild acidic conditions typically
used for ESI- and MALDI-MS, 18O-containing carboxyl groups
of peptides are sufficiently stable (Schnölzer, Jedrzejewski, &
674
Lehmann, 1996). When using 18O labeling of water in the
digestion buffer, cross-linked peptides are readily distinguished
in the mass spectra by a characteristic shift of 8 amu due to the
incorporation of two 18O atoms in each C-terminus. Non-crosslinked, intramolecularly cross-linked peptides as well as peptides
that are modified by a partially hydrolyzed cross-linker contain
one N-terminus and show mass shifts of 4 amu, whereas
intermolecular cross-linked products that contain two N-termini
exhibit a shift of 8 amu compared to their non-labeled counterparts. That interesting approach was exemplified for the
identification of the interacting domains within the heterodimeric
complex of two DNA-repair proteins (Rad18–Rad6) (Back et al.,
2002b) and for probing the tertiary structure of bovine serum
albumin (BSA) (Huang, Kim, & Dass, 2004). In the latter study,
BSA was modified with three homobifunctional, amine-reactive
specific cross-linkers, digested with trypsin, and analyzed by
tandem mass spectrometry.
B. Fluorescence Labeling
A selection of cross-linked products from the complex reaction
mixtures can also be performed using the homobifunctional,
fluorogenic (fluorescence creating) cross-linking reagent
dibromobimane (4,6-dibromomethyl-3,7-dimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione, DBB) (Scheme 3). The
use of DBB in combination with MALDI-TOF-MS and ESI-MS/
MS was demonstrated to identify spatially close cysteines in the
CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES
&
FIGURE 6. Deconvoluted ESI-FTICR mass spectrum of the peptide mixture obtained by in-gel digestion
with a mixture of endoproteinases AspN and LysC from cross-linked LN-domains of laminin b1 and laminin
g1 (2 mM of each protein, 200-fold excess of BS3 over protein concentration) at an incubation time of 15 min.
The magnified insets show the amino acid sequences 1–15 of laminin g1 and 346–370 of laminin b1 that
have been modified by partially hydrolyzed BS3.
eye-lens protein g-crystallin (Sinz & Wang, 2004) and to define
the molecular interfaces between calmodulin and a C-terminal
fragment of the giant muscle protein nebulin (Sinz & Wang,
2001). DBB cross-links thiol pairs that span 3 to 6 Å (Kosower
et al., 1979), is non-fluorescent in solution, and becomes
fluorescent when both of its alkylating groups have reacted
(Kosower & Kosower, 1995). Thus, peptides that contain reacted
DBB can be isolated by HPLC, using fluorescence detection
before mass spectrometric analysis. A major limitation of DBB is
that the unusual fragmentation patterns obtained in MS/MS
experiments are caused by the presence of the incorporated crosslinker (A. Sinz, personal observation).
C. Cleavable Cross-Linkers
Some cross-linkers (Fig. 3D) may be cleaved by periodate in case
they contain a polyol structure (e.g., disuccinimidyl tartrate
(DST)) or by reducing agents, such as dithiothreitol (DTT) in
case they contain a disulfide bond (e.g., 3,30 -dithio-bis(succini-
midylpropionate) (DTSSP)). Alternatively, cross-linkers might
be cleaved during MS/MS experiments in case they contain
bonds that fragment during low-energy activation.
1. Chemical Cleavage
The thiol-cleavable cross-linking reagent DTSSP was applied to
map interfaces of a number of protein complexes (Bennett et al.,
2000; Back et al., 2002a; Davidson & Hilliard, 2003) and to map
the three-dimensional structure of a-crystallin (Peterson, Young,
& Takemoto, 2004). MALDI-TOF-MS and ESI-QTOF-MS were
employed to obtain specific peptide maps of the digested reaction
mixtures before and after reduction, and the respective masses
were compared to each other. Peptides, which were not observed
in the spectrum following reduction, were assigned as putative
cross-links, and cross-links were confirmed on the basis of
observation of the corresponding peptide ‘halves’ of the
previously detected cross-linked products after reduction. This
strategy, however, relies on observing the signals of cross-linking
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TABLE 1. The homobifunctional, amine-reactive, isotope-labeled (d0/d4) cross-linkers
BS2G and BS3 with their respective spacer chain lengths
products before and after reduction, and identification of crosslinked products might be ambiguous in case the corresponding
signals of the peptide ‘halves’ are not observed after reduction.
Moreover, for an assignment of the cross-linked amino acids,
MS/MS experiments are needed.
any useful structural information will be obtained with this
reagent. The presented strategy, however, seems promising and
might serve as basis to design a second generation of crosslinkers with improved properties.
2. MS/MS Cleavage
D. Affinity Cross-Linkers
An amine-reactive, homobifunctional cross-linker N-benzyliminodiacetoyloxysuccinimide (BID) was designed and applied to
cross-linking of model peptides as well as for components of a
protein complex (Back et al., 2001, 2002a). A marker ion (at m/z
91) was readily detected in both triple-quadrupole and QTOF
tandem MS experiments; that tropylium ion indicated the
presence of the incorporated cross-linker.
A novel type of cross-linker named PIR (protein interaction
reporter) was synthesized with two low-energy MS/MS cleavable
bonds in the spacer chain. Two RINK groups that contained a
more-labile bond during low-energy activation compared to
peptide bonds were incorporated into the cross-linker, using
solid-phase chemistry (Tang et al., 2005). The new cross-linker
was used to cross-link ribonuclease S (Rnase S), a non-covalent
complex of S-peptide and S-protein, to release a reporter ion of
m/z 711 that indicated the presence of a cross-linked product.
Limitations of the presented cross-linker consist of a spacer chain
length being 43 Å in its fully extended conformation and in the
reagent’s high flexibility. Therefore, it is questionable whether
Affinity cross-linkers contain a biotin group in addition to the two
reactive groups of a heterobifunctional cross-linker, which
allows enrichment of cross-linker-containing species by affinity
purification on avidin beads (Alley et al., 2000). A modular
approach was developed for the synthesis of trifunctional crosslinking reagents, which were applied to a structural study of the
heterodimeric negative cofactor 2 protein complex (TresterZedlitz et al., 2003). Modularity guarantees a systematic
variation of the reagent, and thus allows the optimization of the
reagent with respect to the desired cross-linking applications.
The reaction of the trifunctional cross-linker sulfo-SBED
(Scheme 2) has been studied, with the peptide neurotensin
(Hurst, Lankford, & Kennel, 2004). In our group, we explored the
applicability of sulfo-SBED to map the interface regions between
calmodulin and its target peptide derived from the skeletal
muscle myosin light-chain kinase (Sinz, Kalkhof, & Ihling,
2005). The cross-linking reaction mixtures were subjected to
tryptic in-solution digestion, and biotinylated peptides (e.g.,
peptides that had been modified by the cross-linker as well as
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CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES
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FIGURE 7. Deconvoluted ESI-FTICR mass spectrum of the tryptic peptide mixture from intramolecularly
cross-linked calmodulin (100-fold excess of BS2G-d0/d4 over protein/peptide concentration) at an
incubation time of 60 min. The magnified insert shows a cross-linked product within the calmodulin
sequence 14–37, in which Lys-21 has reacted with Lys-31. The 1:1 pattern with a mass difference of 4 u
caused by the isotope-labeled cross-linker enhanced the confidence in the assignment of cross-linked
products. Please note that neutral monoisotopic masses are given. Signals that originated from calmodulin
peptides (filled circle), cross-linked products (star), and autolytic peptides of trypsin (diamond) are
indicated. Reprinted from Schmidt et al. (2005) with permission, copyright 2005 IM Publications.
cross-linked peptides) were enriched on monomeric avidin beads
after several washing step. Peptide mixtures were analyzed with
MALDI-TOF-MS and nano-HPLC/nano-ESI-FTICR-MS. One
drawback of sulfo-SBED is that the spacer chain is much longer
(23 Å) than the preferred length of 8 to 15 Å, which is
considered to provide the most useful distance geometry
information about the orientation of lysine residues for threading
calculations (Collins et al., 2003).
A novel trifunctional cross-linker was presented recently,
connecting a biotin moiety via a polyethylene glycol chain to a
homobifunctional NHS ester with a spacer chain length of 9 Å
(Fujii et al., 2004).
SCHEME 3. Chemical structure of the fluorogenic cross-linker
dibromobimane (DBB).
VI. COMPUTER SOFTWARE FOR DATA ANALYSIS
Currently, the greatest deficits of employing chemical crosslinking and MS analysis include the lack of computer software
that can effectively analyze the enormous complexity of the
reaction mixtures. All of the existing programs exhibit their
specific limitations; thus, most of the cross-linked products must
be manually assigned in the mass spectra. Intrapeptide crosslinks or peptides that have been modified by a partially
hydrolyzed cross-linker are readily identified by performing
standard ‘in silico-digestion’ procedures; for example, by using
the ExPASy Proteomics Tool ‘FindPept’ [http://www.expasy.ch].
The mass shift caused by reaction of the peptide with partially
hydrolyzed cross-linker (e.g., in the case of NHS esters) is
defined as a modification. Cross-links between two peptides,
however, are more difficult to identify. Programs that are
currently available as Web server versions for free access
are the Automated Spectrum Assignment Program (ASAP), the
MS2Assign, the MS2PRO software (available at: http://roswell.
ca.sandia.gov/mmyoung/) (Young et al., 2000; Collins et al.,
2003; Kruppa, Schoeninger, & Young, 2003; Schilling et al.,
2003), and the SearchXLinks Version 3.3.3 software (available
at: http://www.searchxlinks.de/cgi-bin/home.pl) (Wefing,
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Schnaible, & Hoffmann, 2001; Schnaible et al., 2002). The
‘Collaboratory for MS3D’ (C-MS3D, available at: http://
ms3d.org/index.php) is a knowledge grid for scientist working
in the field of structural biology. The abbreviation MS3D (mass
spectrometry in three dimensions) has been created recently
(Young et al., 2000) for the strategy of combining chemical
crosslinking with high-resolution mass spectrometry to glean
structural information about proteins and other biological
macromolecules.
ASAP is a tool to assign MS peptide peak lists that have been
generated by chemical cross-linking in a bottom-up approach.
The program calculates the theoretical crosslinking possibilities
for a given protein sequence. Information about the cross-linker,
the protease used for enzymatic digestion as well as potential
post-translational modifications are defined by user input, and
ASAP returns putative assignments for the list of input m/z
values. MS2Assign uses tandem MS peak lists that have been
generated from the fragmentation of cross-linked, modified, or
unmodified peptides in a bottom-up approach (Fig. 1A), whereas
MS2PRO assigns signals that originate from MS/MS spectra of
whole proteins in a top-down approach (Fig. 1B).
SearchXLinks analyzes mass spectra of a modified, crosslinked, and digested protein provided that its amino acid
sequence is known. Apart from searching for intramolecular
cross-linked products, SearchXLinks can be employed to
completely characterize post-translational modifications of a
protein.
A commercially available program that contains a nice
feature to calculate crosslinked products from one or two proteins
is the GPMAW program (General Protein/Mass Analysis for
Windows, Version 6.2.1) (http://welcome.to/gpmaw) (Peri,
Steen, & Pandey, 2001). The user defines one or two protein
sequences of the protein(s) to be cross-linked as well as the crosslinking reagent that was employed (e.g., zero-length, homobifunctional, or heterobifunctional cross-linker). The respective
cross-linker is defined by the amino acids that it targets, its
elemental composition, and the composition of partially hydrolyzed cross-linker (e.g., for NHS esters) or reduced cross-linker
(e.g., for disulfide cleavable cross-linkers).
VII. CONCLUSIONS AND PERSPECTIVES
Structural analysis of proteins by chemical cross-linking
combined with a mass spectrometric analysis of the products is
a rapidly developing area. Recent technological advances in the
field of mass spectrometry are likely to benefit the analysis of the
complex mixtures created by chemical cross-linking. For
confidently assigning cross-linker-containing species, the application of high resolution and high mass accuracy methods is a
valuable prerequisite, therefore, it is likely that FTICR mass
spectrometry will play a much larger role to analyze complex
biological samples in the near future. The application of ECD
FTICR tandem MS for the sequence analysis of cross-linked
proteins and peptides provides high-resolution mass spectrometric data alongside simple fragmentation patterns (Zubarev,
2003). It is possible that a combination of the top-down and
bottom-up approaches will become the most widely used method
678
to characterize cross-linked products. That strategy comprises
the enzymatic or chemical cleavage of a high-molecular weight
cross-linked protein complex into a few smaller pieces that are
subsequently subjected to MS/MS experiments using an FTICR
mass spectrometer.
Enrichment of cross-linker containing species by affinity
purification can greatly reduce the complexity of the mixtures.
One could envision that cross-linkers, which are isotope-labeled
and biotinylated (Trester-Zedlitz et al., 2003), will greatly
facilitate an unambiguous identification of cross-linked products
when large protein assemblies are investigated.
There is still a long way ahead of us before chemical crosslinking combined with mass spectrometry will become a
generally applicable technique for rapid protein structure
characterization. Clearly, improvements are needed and can be
expected in synthesizing novel cross-linking reagents, in better
understanding cross-linker reactivities, in developing innovative
strategies for an enrichment of cross-linked products or
facilitated MS detection, and—probably the most urgent task—
in improving computer software for automated data analysis.
VIII. ABBREVIATIONS
BID
BSA
BS2G
BS3
CE
CID
DBB
DSS
DTSSP
DTT
ECD
EDC
ESI
FTICR
HPLC
IRMPD
MALDI
NHS
PICUP
PIR
QIT
RP
SDS–PAGE
SORI-CID
sulfo-SBED
SWIFT
TOF
N-benzyliminodiacetoyloxysuccinimide
bovine serum albumin
Bis(sulfosuccinimidyl)glutarate
Bis(sulfosuccinimidyl)suberate
capillary electrophoresis
collision-induced dissociation
4,6-dibromomethyl-3,7-dimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione, Dibromobimane
disuccinimidyl suberate
3,30 -dithiobis(sulfosuccinimidylpropionate)
dithiothreitol
electron-capture dissociation
1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide
electrospray ionization
Fourier-transform ion-cyclotron resonance
high-performance liquid chromatography
infrared multi-photon dissociation
matrix-assisted laser desorption/ionization
N-hydroxysuccinimide
photoinduced cross-linking of unmodified
proteins
protein-interaction reporter
quadrupole ion trap
reversed phase
sodium dodecyl sulfate polyacrylamide gel
electrophoresis
sustained off-resonance irradiation collisioninduced dissociation
sulfosuccinimidyl-2-[6-(biotinamido)-2-(pazidobenzamido)-hexanoamido]ethyl-1,30 dithiopropionate)
stored-waveform inverse Fourier transform
time-of-flight
CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES
ACKNOWLEDGMENTS
The junior research group of A.S. is funded by the Saxon State
Ministry of Higher Education, Research and Culture and the
Deutsche Forschungsgemeinschaft (DFG project Si 867/7-1).
Financial support from the Thermo Electron Corporation
(Mattauch-Herzog award of the German Society for Mass
Spectrometry to A.S.) is also gratefully acknowledged. A.S.
thanks Ms. Daniela Schulz and Mr. Stefan Kalkhof for critically
reading the manuscript.
REFERENCES
Alley SC, Ishmael FT, Jones AD, Benkovic SJ. 2000. Mapping proteinprotein interactions in the bacteriophage T4 DNA polymerase
holoenzyme using a novel trifunctional photo-cross-linking and affinity
reagent. J Am Chem Soc 122:6126–6127.
Avrameas S, 1969. Coupling of enzymes to proteins with glutaraldehyde. Use
of the conjugates for the detection of antigens and antibodies.
Immunochemistry 6:43–52.
Back JW, Hartog AF, Dekker HL, Muijsers AO, de Koning LJ, de Jong L.
2001. A new crosslinker for mass spectrometric analysis of the
quaternary structure of protein complexes. J Am Soc Mass Spectrom
12:222–227.
Back JW, Artal Sanz M, de Jong L, de Koning LJ, Nijtmans LGJ, de Koster
CG, Grivell LA, van der Spek H, Muijsers AO. 2002a. A structure for the
yeast prohibitin complex: Structure prediction and evidence from
chemical crosslinking and mass spectrometry. Prot Sci 11:2471–2478.
Back JW, Notenboom V, de Koning LJ, Muijsers AO, Sixma TK, de Koster
CG, de Jong L. 2002b. Identification of cross-linked peptides for protein
interaction studies using mass spectrometry and 18O labeling. Anal
Chem 74:4417–4422.
Back JW, de Jong L, Muijsers AO, de Koster CG. 2003. Chemical crosslinking and mass spectrometry for protein structural modeling. J Mol
Biol 331:303–313.
Bennett KL, Kussmann M, Björk P, Godzwon M, Mikkelsen M, Sørensen P,
Roepstorff P. 2000. Chemical cross-linking with thiol-cleavable
reagents combined with differential mass spectrometric peptide
mapping—A novel approach to assess intermolecular protein contacts.
Protein Sci 9:1503–1518.
Bitan G, Teplow DB. 2004. Rapid photochemical cross-linking—A new tool
for studies of metastable, amyloidogenic protein assemblies. Acc Chem
Res 37:357–364.
Bragg PD, Hou C. 1975. Subunit composition, function, and spatial
arrangement in Ca2þ-activated and Mg2þ-activated adenosine triphosphatases of Escherichia coli and Salmonella typhimurium. Arch
Biochem Biophys 167:311–321.
Brewer CF, Riehm JP. 1967. Evidence for possible nonspecific reactions
between N-ethylmaleimide and proteins. Anal Biochem 18:248.
Browne DT, Kent SBH. 1975. Formation of non-amidine products in reaction
of primary amines with imido esters. Biochem Biophys Res Commun
67:126.
Brunner J. 1993. New photolabeling and cross-linking methods. Annu Rev
Biochem 62:483–514.
Cai K, Itoh Y, Khorana HG. 2001. Mapping of contact sites in complex
formation between transducin and light-activated rhodopsin by covalent
cross-linking: Use of a photoactivatable reagent. Proc Natl Acad Sci
USA 98:4877–4882.
Carlsohn E, Angström J, Emmett MR, Marshall AG, Nilsson CL. 2004.
Chemical cross-linking of the urease complex from Helicobacter pylori
and analysis by Fourier transform ion cyclotron resonance mass
&
spectomretry and molecular modeling. Int J Mass Spectrom 234:137–
144.
Chang Z, Kuchar J, Hausinger RP. 2004. Chemical cross-linking and mass
spectrometric identification of sites of interaction for UreD, UreF, and
Urease. J Biol Chem 279:15305–15313.
Chen X, Chen YH, Anderson VE. 1999. Protein cross-links: Universal
isolation and characterization by isotopic derivatization and electrospray ionization mass spectrometry. Anal Biochem 273:192–203.
Chu BCF, Kramer FR, Orgel LE. 1986. Synthesis of an amplifiable reporter
RNA for bioassays. Nucleic Acids Res 14:5591–5603.
Collins CJ, Schilling B, Young MM, Dollinger G, Guy RK. 2003. Isotopically
labeled crosslinking reagents: Resolution of mass degeneracy in the
identification of cross-linked peptides. Bioorg Med Chem Lett
13:4023–4026.
Comisarov MB, Marshall AG. 1974. Fourier-transform ion-cyclotron
resonance spectroscopy. Chem Phys Lett 25:282–283.
Cuatrecaseas P. 1972. Affinity chromatography of macromolecules. Adv
Enzymol 36:29.
Cuatrecaseas P, Parikh I. 1972. Adsorbents for affinity chromatography—Use
of N-hydroxysuccinimide esters of agarose. Biochemistry 11:2291–
2299.
D’Ambrosio C, Talamo F, Vitale RM, Amodeo P, Tell G, Ferrera L, Scaloni A.
2003. Probing the dimeric structure of porcine aminoacylase 1 by mass
spectrometric and modeling procedures. Biochemistry 42:4430–4443.
Davidson WS, Hilliard GM. 2003. The spatial organization of apolipoprotein
A-1 on the edge of discoidal high density lipoprotein particles. J Biol
Chem 278:27199–27207.
Dihazi GH, Sinz A. 2003. Mapping low-resolution three-dimensional protein
structures using chemical cross-linking and Fourier transform ioncyclotron resonance mass spectrometry. Rapid Commun Mass
Spectrom 17:2005–2014.
Dorman G, Prestwich GD. 1994. Benzophenone photophores in biochemistry. Biochemistry 33:5661–5673.
Dreisewerd K, Berkenkamp S, Leisner A, Rohlfing A, Menzel C. 2001.
Fundamentals of MALDI-MS with pulsed infrared lasers. Int J Mass
Spectrom 226:189–209.
Egnaczyk GF, Greis KD, Stimson ER, Maggio JE. 2001. Photoaffinity crosslinking of Alzheimer’s disease amyloid fibrils reveals interstrand
contact regions between assembled b-amyloid peptide subunits.
Biochemistry 40:11706–11714.
Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. 1989. Electrospray
ionization for mass spectrometry of large biomolecules. Science 46:
64–71.
Friedhoff P. 2005. Mapping protein–protein interactions by bioinformatics
and crosslinking. Anal Bioanal Chem 381:78–80.
Fujii N, Jacobsen RB, Wood NL, Schoeniger JS, Guy RK. 2004. A novel
protein crosslinking reagent for the determination of moderate
resolution protein structures by mass spectrometry (MS3-D). Bioorg
Med Chem Lett 14:427–429.
Füzesi M, Gottschalk KE, Lindzen M, Shainskaya A, Küster B, Garty H,
Karlish SJD. 2005. Covalent cross-links between the g subunit
(FXYD2) and a and b subunits of Na,K-ATPase. Biochemsitry
280:18291–18301.
Gauthier JW, Trautman TR, Jacobson DB. 1991. Sustained off-resonance
irradiation for collision-activated dissociation involving Fourier transform mass spectrometry—Collision-activated dissociation technique
that emulates infrared multiphoton dissociation. Anal Chim Acta
246:211–225.
Gerardi RD, Barnett NW, Lewis SW. 1999. Analytical applications of
Tris(2,20 -bipyridyl)ruthenium(II) as a chemiluminescent reagent. Anal
Chim Acta 378:1–43.
Gershon D. 2003. Proteomics technologies: Probing the proteome. Nature
424:581–587.
679
&
SINZ
Ghosh SS, Kao PM, McCue AW, Chappelle HL. 1990. Use of maleimide-thiol
coupling chemistry for efficient syntheses of oligonucleotide-enzyme
conjugate hybridization probes. Bioconj Chem 1:71–76.
Gilchrist TL, Rees CW. 1969. Carbenes, nitrenes, and arynes, studies in
modern chemistry. London: Nelson Publ. p 131.
Giron-Monzon L, Manelyte L, Ahrends R, Kirsch D, Spengler B, Friedhoff P.
2004. Mapping protein–protein interactions between MutL and MutH
by cross-linking. J Biol Chem 279:49338–49345.
Gorin G, Matic PA, Doughty G. 1966. Kinetics of reaction of Nethylmaleimide with cysteine and some congeners. Arch Biochem
Biophys 115:593.
Green NS, Reisler E, Houk KN. 2001. Quantitative evaluation of the lengths
of homobifunctional protein cross-linking reagents used as molecular
rulers. Protein Sci 10:1293–1304.
Guan S, Marshall AG. 1996. Stored waveform inverse Fourier transform
(SWIFT) ion excitation in trapped-ion mass spectrometry: Theory and
applications. Int J Mass Spectrom Ion Proc 157–158:5–37.
Hartmann FC, Wold F, Bifunctional reagents. 1966. Cross-linking of
pancreatic ribonuclease with a diimido ester. J Am Chem Soc 88:
3890–3891.
Heitz JR, Anderson CD, Anderson BM. 1968. Inactivation of yeast alcohol
dehydrogenase by N-alkylmaleimides. Arch Biochem Biophys 127:
627.
Hermanson GT. 1996. Bioconjugate techniques. San Diego, CA: Academic
Press.
Hoare DG, Koshland DE. 1966. A procedure for selective modification of
carboxyl groups in proteins. J Am Chem Soc 88:2057.
Huang BX, Kim HY, Dass C. 2004. Probing three-dimensional structure of
bovine serum albumin by chemical cross-linking and mass spectrometry. J Am Soc Mass Spectrom 15:1237–1247.
Hunter MJ, Ludwig ML. 1962. Reaction of imidoesters with proteins and
related small molecules. J Am Chem Soc 84:3491.
Hurst GB, Lankford TK, Kennel SJ. 2004. Mass spectrometric detection of
affinity purified crosslinked peptides. J Am Soc Mass Spectrom
15:832–839.
Ihling C, Berger K, Höfliger MM, Führer D, Beck-Sickinger AG, Sinz A.
2003. Nano-high-performance liquid chromatography combined with
nano-electrospray ionization Fourier transform ion-cyclotron resonance mass spectrometry for proteome analysis. Rapid Commun Mass
Spectrom 17:1240–1246.
Itoh Y, Cai K, Khorana HG. 2001. Mapping of contact sites in complex
formation between light-activated rhodopsin and transducin by covalent
crosslinking: Use of a chemically preactivated reagent. Proc Natl Acad
Sci USA 98:4883–4887.
Junge HJ, Rhee JS, Jahn O, Varoqueaux F, Spiess J, Waxham MN,
Rosenmund C, Brose N. 2004. Calmodulin and Munc13 form a Ca2þ
sensor/effector complex that controls short-term synaptic plasticity.
Cell 118:389–401.
Kalkhof S, Ihling C, Mechtler K, Sinz A. 2005. Chemical cross-linking and
high performance Fourier transform ion cyclotron resonance mass
spectrometry for protein interaction analysis: Application to a
calmodulin/target peptide complex. Anal Chem 77:495–503.
Karas M, Hillenkamp F. 1988. Laser desorption ionization of proteins with
molecular masses exceeding 10,000 Da. Anal Chem 60:2299–2301.
Karas M, Krüger R. 2003. Ion formation in MALDI: the cluster ionization
mechanism. Chem Rev 2003 103:427–439.
Kelleher NL, Lin HY, Valsakovic GA, Aaserud DJ, Fridrikson EK, Beavil A,
Holowka D, Gould HJ, Baird B, McLafferty FW. 1999. ‘Top down’
versus ‘bottom up’ protein characterization by tandem high-resolution
mass spectrometry. J Am Chem Soc 121:806–812.
Kiehm D, Ji TH. 1977. Photochemical cross-linking of cell membranes—Test
for natural and random collisional cross-links by millisecond crosslinking. J Biol Chem 252:8524–8531.
680
Kluger R, Alagic A. 2004. Chemical cross-linking and protein–protein
interactions—A review with illustrative protocols. Bioorg Chem
32:451–472.
Kodadek T, Duroux-Richard I, Bonnafous JC. 2005. Techniques: Oxidative
cross-linking as an emergent tool for the analysis of receptor-mediated
signaling events. Trends Pharmacol Sci 26:210–217.
Kosower EM, Kosower NS. 1995. Bromobimane probes for thiols. Methods
Enzymol 251:133–148.
Kosower NS, Kosower EM, Newton GL, Ranney HM. 1979. Bimane
fluorescent labels: Labeling of normal human red cells under
physiological conditions. Proc Natl Acad Sci USA 76:3382–3386.
Kruppa GH, Schoeninger JS, Young MM. 2003. A top down approach to
protein structural studies using chemical cross-linking and Fourier
transform mass spectrometry. Rapid Commun Mass Spectrom 17:155–
162.
Lanman J, Lam TT, Barnes S, Sakalian M, Emmett MR, Marshall AG,
Prevelige PE. 2003. Identification of novel interactions in HIV-1 capsid
protein assembly by high resolution mass spectrometry. J Mol Biol
325:759–772.
Leavell MD, Novak P, Behrens CR, Schoeniger JS, Kruppa GH. 2004.
Strategy for selective chemical cross-linking of tyrosine and lysine
residues. J Am Soc Mass Spectrom 15:1604–1611.
Little DP, Speir JP, Senko MW, O’Connor PB, McLafferty FW. 1994. Infrared
multiphoton dissociation of large multiply charged ions for biomolecule
sequencing. Anal Chem 66:2809–2815.
Liu SC, Fairbanks G, Palek J. 1977. Spontaneous, reversible protein crosslinking in human erythrocyte membrane—Temperature and pHdependence. Biochemistry 16:4066.
Lomant AJ, Fairbanks G. 1976. Chemical probes of extended biological
structures—Synthesis and properties of cleavable protein cross-linking
reagent dithiobis(succinimidyl-S-35 propionate). J Mol Biol 104:243–
261.
Marshall AG. 2000. Milestones in Fourier transform ion cyclotron resonance
spectrometry technique development. Int J Mass Spectrom 200:331–
356.
McLafferty FW, Fridriksson EK, Horn DM, Lewis MA, Zubarev RA. 1999.
Biochemistry—Biomolecule mass spectrometry. Science 284:1289–
1290.
Melcher K. 2004. New chemical crosslinking methods for the identification of
transient protein–protein interactions with multiprotein complexes.
Curr Prot Pept Sci 5:287–296.
Menzel C, Dreisewerd K, Berkenkamp S, Hillenkamp F. 2001. Mechanisms
of energy deposition in infrared MALDI-MS. Int J Mass Spectrom
207:73–96.
Müller DR, Schindler P, Towbin H, Wirth U, Voshol H, Hoving S, Steinmetz
MO. 2001. Isotope-tagged cross-linking reagents. A new tool in mass
spectrometric protein interaction analysis. Anal Chem 73:1927–1934.
Novak P, Young MM, Schoeninger JS, Kruppa GH. 2003. A top-down
approach to protein structure studies using chemical cross-linking and
Fourier transform mass spectrometry. Eur J Mass Spectrom 9:623–631.
Novak P, Haskins WE, Ayson MJ, Jacobsen RB, Schoeniger JS, Leavell MD,
Young MM, Kruppa GH. 2005. Unambiguous assignment of intramolecular chemical cross-links in modified mammalian membrane
proteins by Fourier transform-tandem mass spectrometry. Anal Chem
77:5101–5108.
Onisko B, Guitian Fernandez E, Louro Freire M, Schwarz A, Baier M,
Camina F, Rodriguez Garcia J, Rodriguez-Segade Villamarin S,
Requena JR. 2005. Probing PrPSC structure using chemical crosslinking and mass spectrometry: Evidence of the proximity of Gly90
amino termini in the PrP 27–30 aggregate. Biochemistry 44:10100–
10109.
Partis MD, Griffiths DG, Roberts GC, Beechey RB. 1983. Cross-linking of
protein by omega-maleimido alkanoyl N-hydroxysuccinimido esters. J
Prot Chem 2:263–277.
CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES
Pearson KM, Pannell LK, Fales HM. 2002. Intramolecular cross-linking
experiments on cytochrome-c and ribonuclease A using an isotope
multiplet method. Rapid Commun Mass Spectrom 16:149–159.
Peri S, Steen H, Pandey A. 2001. GPMAW—A software tool for analyzing
proteins and peptides. Trends Biochem Sci 26:687–689.
Peterson JJ, Young MM, Takemoto LJ. 2004. Probing a-crystallin structure
using chemical cross-linkers and mass spectrometry. Molecul Vision
10:857–866.
Petrotchenko EV, Pedersen LC, Borchers CH, Tomer KB, Negishi M. 2001.
The dimerization motif of cytosolic sulfotransferases. FEBS Lett
490:39–42.
Pierce Perbio. 2003/2004. Applications handbook and catalog, Rockford, IL.
Prescher JA, Bertozzi CR. 2005. Chemistry in living systems. Nat Chem Biol
1:13–21.
Rappsilber J, Siniossoglou S, Hurt EC, Mann M. 2000. A generic strategy to
analyze the spatial organization of multi-protein complexes by crosslinking and mass spectrometry. Anal Chem 72:267–275.
Schilling B, Row RH, Gibson BW, Guo X, Young MM. 2003. MS2Assign,
automated assignment and nomenclature of tandem mass spectra of
chemically crosslinked peptides. J Am Soc Mass Spectrom 14:834–
850.
Schmidt A, Kalkhof S, Ihling C, Cooper DMF, Sinz A. 2005. Mapping protein
interfaces by chemical cross-linking and fticr mass spectrometry:
Application to a calmodulin/adenylyl cyclase 8 peptide complex. Eur J
Mass Spectrom 11:525–534.
Schnaible V, Wefing S, Resemann A, Suckau D, Bücker A, Wolf-Kümmeth S,
Hoffmann D. 2002. Screening for disulfide bonds in proteins by MALDI
in-source decay and LIFT-TOF/TOF-MS. Anal Chem 74:4980–4988.
Schnölzer M, Jedrzejewski P, Lehmann WD. 1996. Protease-catalyzed
incorporation of O-18 into peptide fragments and its application for
protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 17:945–953.
Schulz DM, Ihling C, Clore GM, Sinz A. 2004. Mapping the topology and
determination of a low-resolution three-dimensional structure of the
calmodulin-melittin complex by chemical cross-linking and highresolution FTICR-MS: Direct demonstration of multiple binding
modes. Biochemistry 43:4703–4715.
Shen Y, Zhao R, Belov ME, Conrads TP, Anderson GA, Tang K, Pasa-Tolic L,
Veenstra TD, Lipton MS, Smith RD. 2001. Packed capillary reversedphase liquid chromatography with high-performance electrospray
ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics. Anal Chem 73:1766–1775.
Silva RAGD, Hilliard GM, Fang J, Macha S, Davidson WS. 2005. A threedimensional molecular model of lipid-free apolipoproteins A-I
determined by cross-linking/mass spectrometry and sequence threading. Biochemistry 44:2759–2769.
Sinz A. 2003. Chemical cross-linking and mass spectrometry for mapping
three-dimensional structures of proteins and protein complexes. J Mass
Spectrom 38:1225–1237.
Sinz A. 2005. Chemical cross-linking and FTICR mass spectrometry for
protein structure characterization. Anal Bioanal Chem 381:44–47.
Sinz A, Kalkhof S, Ihling C. 2005. Mapping protein interfaces by a
trifunctional cross-linker combined with MALDI-TOF and ESI-FTICR
mass spectrometry. J Am Soc Mass Spectrom 16:1921–1931.
Sinz A, Wang K. 2001. Mapping protein interfaces with a fluorogenic crosslinker and mass spectrometry: Application to nebulin–calmodulin
complexes. Biochemistry 40:7903–7913.
Sinz A, Wang K. 2004. Mapping spatial proximities of sulfhydryl groups in
proteins using a fluorogenic cross-linker and mass spectrometry. Anal
Biochem 331:27–32.
Smyth DG, Konigsberg W, Blumenfeld OO. 1964. Reactions of Nethylmaleimide with peptides and amino acids. Biochem J 91:589.
&
Staros JV. 1988. Membrane-impermeant cross-linking reagents—Probes of
the structure and dynamics of membrane proteins. Acc Chem Res
21:435–441.
Staros JV, Wright RW, Swingle DM. 1986. Enhancement by N-hydroxysulfosucccinimide of water-soluble cabodiimide-mediated coupling
reactions. Anal Biochem 156:220–222.
Suchanek M, Radzikowska A, Thiele C. 2005. Photo-leucine and photomethionin allow identification of protein–protein interactions in living
cells. Nature Meth 2:261–267.
Swaim CL, Smith JB, Smith DL. 2004. Unexpected products from the
reaction of the synthetic cross-linker 3,30 -dithiobis(sulfosuccinimidyl
propionate), DTSSP with peptides. J Am Soc Mass Spectrom 15:736–
749.
Tang X, Munske GR, Siems WF, Bruce JE. 2005. Mass spectrometry
identifiable cross-linking strategy for studying protein–protein interactions. Anal Chem 77:311–318.
Taverner T, Hall NE, O’Hair RAJ, Simpson RJ. 2002. Characterization of an
antagonist interleukin-6 dimer by stable isotope labeling, cross-linking,
and mass spectrometry. J Biol Chem 277:46487–46492.
Trakselis MA, Alley SC, Ishmael FT. 2005. Identification and mapping of
protein–protein interactions by a combination of cross-linking,
cleavage, and proteomics. Bioconjug Chem 16:741–750.
Trester-Zedlitz M, Kamada K, Burley SK, Fenyö D, Chait BT, Muir TW.
2003. A modular cross-linking approach for exploring protein
interactions. J Am Chem Soc 125:2416–2425.
Van Dijk ADJ, Boelens R, Bonvin AMJJ. 2005. Data-driven docking for the
study of biomolecular complexes. FEBS J 272:293–312.
Vasilescu J, Guo X, Kast J. 2004. Identification of protein–protein
interactions using in vivo cross-linking and mass spectrometry.
Proteomics 4:3845–3854.
Wefing S, Schnaible V, Hoffmann D. 2001. SearchXLinks, http://
www.searchxlinks.de/, center of advanced european studies and
research (caesar), Bonn, Germany.
Wilbur DS. 1992. Radiohalogenation of proteins—An overview of radionuclides, labeling methods, and reagents for conjugate labeling.
Bioconj Chem 3:433–470.
Wilm M, Mann M. 1994. Electrospray and Taylor-Cone theory, Dole’s Beam
of macromolecules at last? Int J Mass Spectrom Ion Proc 136:167–
180.
Wilm M, Mann M. 1996. Analytical properties of the NanoESI source. Anal
Chem 68:1–8.
Wine RN, Dial JM, Tomer KB, Borchers CH. 2002. Identification of
components of protein complexes using a fluorescent photo-crosslinker and mass spectrometry. Anal Chem 74:1939–1945.
Wong SS. 1991. Chemistry of protein conjugation and cross-linking. Boca
Raton: CRC Press Inc.
Yergey AL, Coorssen JR, Backlund PS, Blank PS, Humphrey GA,
Zimmerberg J, Campbell JM, Vestal ML. 2002. De novo sequencing
of peptides using MALDITOF/TOF. J Am Soc Mass Spectrom 13:784–
791.
Young MM, Tang N, Hempel JC, Oshiro CM, Taylor EW, Kuntz ID, Gibson
BW, Dollinger D. 2000. High throughput protein fold identification by
using experimental constraints derived from intramolecular cross-links
and mass spectrometry. Proc Natl Acad Sci USA 97:5802–5806.
Zenobi R, Knochenmuss R. 1999. Ion formation in MALDI-MS. Mass
Spectrom Rev 17:337–366.
Zubarev RA. 2003. Reactions of polypeptide ions with electrons in the
gasphase. Mass Spectrom Rev 22:57–77.
Zubarev RA, Kelleher NL, McLafferty FW. 1998. Electron capture
dissociation of multiply charged protein cations. A nonergodic process.
J Am Chem Soc 120:3265–3266.
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Andrea Sinz received her degree in Pharmacy from the University of Tübingen (Germany) in
1993. She received her PhD in Pharmaceutical Chemistry from the University of Marburg
(Germany) in 1997. From 1998 to 2000 she served as a postdoctoral fellow at the National
Institutes of Health in Bethesda, MD. Since 2001, she is head of the junior research group
‘Protein-Ligand Interaction by Ion Cyclotron Resonance Mass Spectrometry’ at the
Biotechnological-Biomedical Center in the Department of Chemistry at the University of
Leipzig (Germany).
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