Three-dimensional Protein Structures

7/25/2019 Three-dimensional Protein Structures

1/20

CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY TOMAP THREE-DIMENSIONAL PROTEIN STRUCTURES ANDPROTEINPROTEIN 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 thefunctionof a protein is theanalysis

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. Thelocation 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

proteinshas 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., MassSpec Rev 25:663682, 2006Keywords: chemical cross-linking; bottom-up approach; top-

down 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 numberof identified proteins has dramatically increased during the past

few years. The physiological function of many of the newlydiscovered proteins, however, remains unclear. Closely related to

the study of the function of a protein is the analysis of its three-dimensional structure and the identification of its interactionpartners. In those cases where high-resolution methods for

structural analysis are applicable, such as X-ray crystallography

and NMR spectroscopy, the solved three-dimensional structureof a protein gives insights into stable interactions within a proteincomplex. Theoretical modeling might reveal further interactions,using the known three-dimensional structures as a starting point.

An alternative approachis to build up a set of structurally defined

interactions by covalently connecting pairs of functional groupswithin a protein or a proteincomplex. The location of the createdcross-links imposes a distance constraint on the location ofthe respective side chains and allows drawing conclusions on thedistance 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 theproteolytic peptides that are analyzed (in case a bottom-upstrategy is employed). (2) Analysis is generally fast and, infavorable circumstances, requires only femtomole amounts oftotal protein. (3) It is possible to gain insights into three-dimensional structuresof proteins in solution andflexible regions

are readily identified. (4) Membrane proteins and proteins thatexist as mixtures of different species (post-translational mod-ifications, splicevariants) are amenableto analysis.(5) The broad

range of specificities available for crosslinking reagents towardscertainfunctional groups, such as primaryamines, sulfhydryls,orcarboxylic acids, and the wide range of distances that differentcross-linking reagents can bridge, offer the possibility to performa wide variety of experiments (Hermanson, 1996). Whenselecting a specific reagent with a certain spacer length, oneshould be aware that the average span of a cross-linker can be less

than the maximum calculated distance, according to stochasticdynamics calculations (Green, Reisler, & Houk, 2001).

However, despite the straightforwardness of the cross-linking approach, the identification of the cross-linked productscan be quite cumbersome due to the complexity of the reactionmixtures. Several strategies have been employed to enrich cross-linker-containing species by affinity chromatography or tofacilitate the identification of the cross-linked products; forexample 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 applicationprotocols (Brunner, 1993; Kluger & Alagic, 2004; Melcher,

2004; Kodadek, Duroux-Richard, & Bonnafous, 2005) or on theidentification of protein protein interactions, using chemical

Mass Spectrometry Reviews, 2006, 25, 663 682

# 2006 by Wiley Periodicals, Inc.

Contract grant sponsor: Saxon State Ministry of Higher Education,

Research and Culture; Contract grant sponsor: Deutsche Forschungs-

gemeinschaft (DFG project Si 867/7-1).

*Correspondence to: Dr. Andrea Sinz, Biotechnological-Biomedical

Center, Faculty of Chemistry and Mineralogy, University of Leipzig,

Linnestrasse 3, D-04103 Leipzig, Germany.

E-mail: [email protected]


2/20

cross-linking combined with a mass spectrometric analysis of thecross-linked products (Back et al., 2003; Sinz, 2003; Friedhoff,2005; Trakselis, Alley, & Ishmael, 2005). The issue of using theobtained information as a basis to create structural models of theprotein complexes has been addressed as well (Van Dijk,Boelens, & Bonvin, 2005) and cross-linking combined with

mass spectrometry has been used to distinguish between thecrystallographic and physiological interface in dimeric proteins(Petrotchenko et al., 2001).

In this review article, the most popular cross-linkingreagents for protein structure analysis are described and anoverviewis given on the different availablestrategies that employchemical cross-linking and mass spectrometry. This articlehighlights the challenges for mass spectrometry caused by theenormous complexity of cross-linking reaction mixtures. Thus,

the various approaches described in the literature to facilitatemass spectrometric detection of cross-linked products as well ascomputer software for data analysis are reviewed. The presentreview is not intended to give a comprehensive overview of the

dramatically increasing number of reports on the analysis ofprotein structures, using well-established cross-linkers and MSmethods, but it aims to give insight into novel and promisingstrategies. Application of those novel strategies to real-lifeproblems will show those proposed methods that will prevail.

II. CROSS-LINKING STRATEGIES

To conduct chemical cross-linking experiments of proteins, twoalternative strategies exist in principle, which is commonlyreferred to as bottom-up and top-down approaches. In recentlydeveloped innovative strategies, cross-linking reactions are

conducted in living cells by directly incorporating reactivegroups into the protein, using the cells own biosyntheticmachinery. In the following paragraph, the three strategies willbe described and compared to each other with respect to thestrengths and limitations of each strategy.

A. Bottom-Up Approach

In the bottom-up approach, the protein reaction mixture isenzymatically digested after the cross-linking reaction, and massspectrometric 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 todetermine low-resolution three-dimensional structures of pro-

teins (Young et al., 2000; Sinz, 2003). The most importantprerequisite to successfully conduct crosslinking experiments isa detailed description of the respective amino acid sequences ofthe proteins under investigation. Full sequence coverage shouldbe envisioned to fully characterize the protein with respect to

possible amino acid variants, post-translational modifications, orsplicevariants. When conducting cross-linking reactions, controlsamples must be included, to which no cross-linker is added, toexclude 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,

while not disrupting the three-dimensional protein structures byintroducing too many cross-links per molecule.

After the cross-linking reaction, one-dimensional gelelectrophoresis (SDS PAGE) and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) MS analysisof the reaction mixture can be used to check for the extent of

cross-linked product formation and to optimize the reactionconditions. After the cross-linking reaction, there are severalways to isolate the cross-linked proteins from the reaction

mixture. If SDS PAGE of the cross-linking reaction mixture isperformed, the band of the cross-linked protein or the cross-linkedprotein complex is excised from the 1D-gel and subjectedto enzymatic in-geldigestion (Fig. 1A). Based on the stainingintensity of the respective gel bands, the amount of cross-linkedproduct formation is approximated. Alternatively, the cross-

linked protein or protein complex is separated from the reactionmixture by size-exclusion chromatography, and the digestion isperformed in the solution (Fig.1A).We foundthat an in-solutiondigestion of the cross-linked proteins is far more efficient than

in-gel digestion, where80%of theproteinis lost. Theresultinghighly complex peptide mixtures generated from enzymaticdigestion contained unmodified peptides of the protein(s),peptides modified by partially hydrolyzed cross-linker, andintramolecular (inter- and intra-peptide) cross-linked products

between peptides originating from one protein as well asintermolecular cross-linked products between peptides fromdifferent proteins (Fig. 1A). Peptide mixtures that originatedfrom proteolytic digestion of cross-linking reaction mixtureswere analyzed by MALDI or ESI mass spectrometry. The cross-

linked peptides were assigned in the mass spectra, usingcustomized 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 non-cross-linked proteins, cross-linked products were identified toultimately provide further information on the spatialdistances between functional groups of the protein(s) underinvestigation.

One of the inherent problems of the bottom-up strategy isthat large peptides are commonly created from cross-linkedproteins during enzymatic proteolysis due to a high frequency ofmissed cleavages. Missed cleavages occur because the most

commonly employed cross-linking reagents react with primaryamine groupsat lysine residues and theN-termini of proteins,andtrypsinthe most commonly used proteolytic enzymewill notcleave C-terminal to a modified lysine residue. Another

limitation of the bottom-up approach is that cross-linkedproducts with low charge states are frequently created during

electrospray ionization due to a loss of positive charge aftermodification of the e-amino groups of lysine residues; thatmodification 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 risingnumber of amino acid residues in the peptide. Thus, massspectrometric techniques, which yield high resolution and highmass accuracy data, and moreover allow fragmentation of largepeptides in MS/MS experiments, are a critically importantprerequisite for an unambiguous assignment of cross-linked

products.

& SINZ

664


3/20

B. Top-Down Approach

One of the most direct techniques to analyze cross-linkedproducts is the top-down approach, in which the cross-linkedproteins are analyzed intact rather than being digested before themass spectrometric analysis (McLafferty et al., 1999; Kelleheret al., 1999) (Fig. 1B). Electrospray ionization-Fourier-transform

ion-cyclotron resonance (ESI-FTICR) mass spectrometry is themethod of choice for this kind of analysis. So far, the top-downapproach has been exclusively employed to determine low-resolution three-dimensional structures of proteins from intra-

molecular cross-linking experiments (Kruppa, Schoeninger, &Young, 2003; Novak et al., 2003; 2005). The cross-linkingreaction mixture is presented to the FTICR mass spectrometer,and the cross-linked product is isolated in the ICR cell before it isinterrogated 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 toselect and fragment ions prior to the ICR cell. Determination ofthe accurate mass of the intact cross-linked product provideshints on the number of incorporated cross-linker molecules as

well as on the number of modifications caused by partiallyhydrolyzed cross-linkers. The top-down approach presents someadvantages over the bottom-up approach in that it eliminates the

need to separate the reacted protein from the cross-linkingreaction mixture before the mass spectrometric analysis, becausethis separation is accomplished by a gas-phase purification inthe mass spectrometer. After fragmentation of the cross-linkedproteinin the FTICR mass spectrometer, assignment of the cross-

linked products is performed manually or by customizedsoftware programs (MS2PRO, available at: http://roswell.ca.-sandia.gov/mmyoung/). Top-down approaches have been

successfully employed to assign intramolecular crosslinkedproducts 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 withFTICR-MS because it allows a comprehensive fragmentation oflarge peptides, while post-translational modifications are keptintact.One limitationof thetop-down approach is that analyses of

large protein assemblies are difficult to perform. In the case ofcharacterizing bovine rhodopsin (Novak et al., 2005), the proteinwas proteolyzed into large peptide fragments, using cyanogenbromide, which cleaves at the C-terminal site of methionineresidues, before ESI-FTICR-MS/MS experiments were con-

ducted in a top-down fashion. That combination of bottom-up

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.

CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY FOR PROTEIN STUDIES &

665


4/20

and top-down analysis will most likely become the strategy withthe greatest potential for a rapid and efficient analysis of a widevariety of cross-linking reaction mixtures.

C. Chemical Cross-Linking in Living Systems

Very recently, several highly appealing approaches have beendeveloped, which permit cross-linking of interacting proteins intheir 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 andgenerates cross-links between interacting proteins in thecell. Proteins that are cross-linked to a myc-tagged protein ofinterest are co-purified by immunoaffinity chromatography,

the cross-linked complexes are subsequently dissociated, thebound proteins are separated by one-dimensional gel electro-phoresis and identified by tandem MS (Vasilescu, Guo, & Kast,2004).

Anotherintriguing strategy is based on theincorporation of aunique chemical into a protein of interest, using the cells ownbiosyntheticmachinery (Prescher & Bertozzi, 2005),followedbya chemical reaction with a small-molecule probe. So far, only ahandful of chemical motifs are known to possess the requisitequalities of biocompatibility and selective reactivity to function

as chemical reporters in living cells. Such chemical motifsinclude the tetra-Cys motif that reacts selectively with biarseni-cals or azides that react with phosphines in a Staudinger ligationor in a copper-catalyzed click chemistry type reaction with

alkynes (Prescher & Bertozzi, 2005). In another interestingstudy, three new photoactivatable amino acids that contain adiazirine moiety were designed and termed photo-methionineand photo-leucine, and photo-isoleucine (Suchanek,Radzikowska, & Thiele, 2005) (Fig. 2). The structural similarity

to the natural amino acids methionine, leucine, and isoleucineallows the artificial amino acids to escape the stringent identitycontrol mechanisms of a cell during protein synthesis and to beincorporated into proteins by the cells translation machinery.

Activation by UV light induces covalent cross-linking (seeparagraph photoreactive cross-linkers). The preference formethionine, leucine, and isoleucine implies that transmembranedomains as well as hydrophobic contact areas between proteinsare preferentially cross-linked. In this preference, the method is

complementary to the most commonly employed amine-reactivechemical 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 notbeen 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 betweendifferent molecules (intermolecular) or within parts of amolecule (intramolecular). The hundreds of reagents described

in the literature (Wong, 1991; Hermanson, 1996) or offered

commercially are based on a small number of organic chemicalreactions, thus reducing the number of functionalities in theprotein that can react with the cross-linker. In the following

paragraph, the most widely used classes of cross-linking reagentsare described with respect to their specific strengths andlimitations.

1. Amine-Reactive Cross-Linkers

a. N-hydroxysuccinimide esters. N-hydroxysuccinimide(NHS) esters are probably the most widely applied principleto create reactive acylating reagents. Thirty years ago, NHS

esters were introduced as homobifunctional, highly amine-reactive, cross-linking reagents (Bragg & Hou, 1975; Lomant &Fairbanks, 1976). Because many NHS esters are insoluble inaqueous buffers, most protocols involve dissolving the NHS

esterin an organic solvent at high concentration anddiluting thatstock solution in the reaction medium. Alternatively, sulfo-NHSestersthe water-soluble analogsare used. NHS esters react

with nucleophiles to release the NHS or sulfo-NHS group andtocreate stable amide and imide bonds with primary or secondary

amines, such as freeN-terminus ande-amino groups in lysineside chains of proteins (Scheme 1A). NHS esters exhibit half-lifes in theorder of hours under physiological pH conditions (pH7.07.5) with hydrolysis and amine reactivity increasing whenthe pH is raised (Cuatrecaseas, 1972; Lomant & Fairbanks,

1976; Staros, 1988; Hermanson, 1996). By varying the molarratio of cross-linking reagent and protein the level ofmodification is adapted to the requirements of the individualapplication.

According to the older literature (Hermanson, 1996), thereaction of NHS esters with sulfhydryl groups (in cysteines) or

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).

& SINZ

666


5/20

hydroxyl groups(e.g.,in serineor threonine)does notyield stableproducts because the thioester or ester products hydrolyzerapidly in aqueous solution. Imidazole nitrogens of the histidinering might also be acylated by NHS esters, but the reactionproducts are also rapidly hydrolyzed (Cuatrecaseas & Parikh,1972). Based on our own experience with NHS esters, however,

we frequently observed cross-linked products of serine hydroxylgroups in MS analysis, so the esters were apparently sufficientlystable in aqueous solution. This observation was confirmed by a

carefully conducted study, using the homobifunctional, amine-reactive, and cleavable cross-linker DTSSP (3,30-dithiobis[sul-fosuccinimidyl propionate]) (Swaim, Smith, & Smith, 2004).Reaction products of several model peptides with DTSSP wereanalyzed by ESI-QqTOF mass spectrometry and reactionproducts were confirmed by tandem MS experiments. The NHS

ester DTSSP was found to react unexpectedly with contaminantammonium ions in the buffer solution and with serine andtyrosine residues in addition to the desired reactions with lysineresidues and the N-terminus (Swaim, Smith, & Smith, 2004).

Another study describes the formation of stable products whenNHS esters reacted with primary amines and tyrosine OH groups(Leavell et al., 2004). Under acidic conditions (pH 6.0), the NHSesters were found to react preferentially with theN-terminus andtyrosine hydroxyl groups; however, under alkaline conditions(pH 8.4) they were found to react preferentially with the N-

terminus and lysine amine groups. These findings underline theurgentneed to conduct further research on thereactivities of NHSesters, and cross-linking reagents in general; those studies haveunfortunately been largely neglected so far.

b. Imidoesters. The imidate functional group is one of themost specific acylating groups to modify primary amines, and

cross-linking reagents that contain imidoesters at both endsare among the oldest reagents used for protein conjugation(Scheme 1B) (Hartmann & Wold, 1966). Unlike most othercross-linking reagents, imidoesters possess minimal crossreactivity towards other nucleophiles. The N-terminus of theprotein as well as thee-amino groups in lysine side chains react

best at a pH between 8 and 9. Imidoesters are highly water-soluble, but undergo continuous degradation due to hydrolysis,which reduces the half-life of the imidate group to less than30 min (Hunter & Ludwig, 1962; Browne & Kent, 1975). Thereaction 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 thepositive charge of the lysine group presents one of the major

advantages of imidoesters because one of the greatest dangers todistort the three-dimensional structure of proteins arises fromremoving the positive charge at the lysine residues when aminogroups are targeted in cross-linking reactions. From ourexperience, using imidoesters for cross-linking of proteins atphysiological 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 aminegroups are targeted.

c. Carbodiimides. Carbodiimides are so-called zero-lengthcross-linking reagents because they do not introduce a spacer

chain into the protein. They are used to mediate amide bondformation between spatially close (


6/20

however, are that they are activated by short-wavelength UVirradiation (


7/20

that can insert into a heteroatom-H or CH bond (reactionScheme1F). Unfortunately, photolysis of diazirinesmightlead todiazo isomers, which present strongly alkylating species that are

responsible for undesired reactions in the dark (Brunner, 1993).

c. Benzophenones. A completely different photochemistrycompared to aryl azides or diazirines is exhibited by benzophe-

nones (Dorman & Prestwich, 1994; Egnaczyk et al., 2001; Jungeet 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 alkylradicals created react by forming a new CC bond between the

photophor and the receptor protein. In contrast to diazirinecompounds, activation of benzophenones does not proceedaccording to a photo-dissociative mechanism and is, therefore,reversible.

d. Photo-induced cross-linking of unmodified proteins

(PICUP). Recently, oxidative crosslinking techniques havebeen developed that are mediated by high-valent metal chelate

complexes. The main advantage of that type of reaction is thatthey are extremely rapid and do not require a chemicalmodification of the protein (Kodadek, Duroux-Richard, &

Bonnafous, 2005). The high-valent metal complexes employedare derived from stable, lower-valent species, such as His6-Ni2,Gly-Gly-His-Ni2, or tris(2,20-bipyridyl)ruthenium (II) dication

([Ru(bipy)3]2) (Kodadek, Duroux-Richard, & Bonnafous,

2005). Light irradiation of the water-soluble ruthenium complex[Ru(bipy)3]

2 in the presence of single-electron acceptorsmediates fast and high-yield cross-linked product formation ofproteins. That process has been termed Photo-induced Cross-Linking 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) isoxidized to Ru(III). Ru(III) is a strong one-electron oxidizer thatabstracts 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 polypeptidechain; however, the radical will form preferentially at side chainsthat offer stabilization through aromatic or neighboring groupeffects, such as histidine, tyrosine, or cysteine residues. Once the

protein radical is created, it can attack a neighboring protein tocreate an intermolecular cross-link (Scheme 1H). One of themajor advantages of the PICUP method is that the reactionrequires very short time period (less than 1 sec) and that it occursacross a wide pH range. The cross-linking efficiency is usually

SCHEME 1. (Continued)


669


8/20

high and the ability of the reagent to function as a zero-lengthcross-linker makes it an attractive tool to take snapshots ofdynamic protein associations. The PICUP strategy has beenemployed to study the structure, kinetics, and thermodynamics ofthe formation of metastable protein oligomers. Those solubleoligomers are precursors for amyloid fibrils that are the primary

toxic effectors responsible for the disease process in amyloi-doses, such as Alzheimers disease (Bitan & Teplow, 2004).

B. Cross-Linker Design

1. Homobifunctional Cross-Linkers

Homobifunctional cross-linking reagents contain identical func-

tional groups at both reactive sites, which are connected with acarbon-chain spacer that bridges a defined distance (Fig. 3A) andthus, allows identical functionalgroups of proteins (e.g.,amine or

sulfhydryl groups) to be cross-linked. The variety of homo-bifunctional cross-linking reagents has increased dramaticallyduring the past 25 years, and today, a wide variety of reagents arecommercially available that possess different spacer lengthsand reactivities (product catalogs available, e.g., at http://www.piercenet.com/ and http://www.trc-canada.com/).

The main disadvantage of homobifunctional reagents is theirsusceptibility to create a wide range of poorly defined products(Avrameas, 1969). The cross-linking reagent reacts initially witha protein molecule to form an intermediate, which could react

with a second protein molecule to create a high-molecular weightaggregate, or which alternatively, could react intramolecularlywith a neighboring functional group on the same polypeptidechain. Maintaining a protein concentration in the mM range

during the reaction is generally desirable in that it reducesunwanted intermolecular cross-linking between proteins. Tocheck for high-molecular weight aggregates due to intermole-cular cross-linking, one-dimensional gel electrophoresis as wellas a rapid mass spectrometric screening, for example, byMALDI-TOF-MS should be performed to establish the optimum

cross-linking reaction conditions for the different cross-linkingreagents. Special caution has to be applied not to disturb thethree-dimensional structure of the proteins by excessive cross-linking, but on the other hand, sufficient amounts of cross-linking

products have to be created to allow for a subsequent massspectrometric detection.

Especially single-step reaction procedures, using homo-bifunctional cross-linking reagents, in which all reagents are

added at the same time to the reaction mixture, pose the greatestpotential to form a multitude of different cross-linked products.To overcome that limitation, two-step protocols have beendeveloped, in which one of the proteins is first reacted with thecross-linker to form an activated protein. After the reaction,

excess reagent is removedand the activated protein is mixed withthe 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

proteins; for example, an amine and a sulfhydryl group(Fig. 3B). Those cross-linking reagents are used to cross-linkproteins favorably in two- or three-step protocols to minimize the

degree of high-molecular weight aggregate formation. Forexample, an NHS ester/maleimide heterobifunctional cross-linker can be applied for reaction with a proteinamine group at itsNHS ester function, whereas the maleimide group does not reactdue to its higher stability in aqueous solution. After a purificationstep, the maleimide function of the cross-linker can react with a

protein sulfhydryl group. Heterobifunctional cross-linkers thatcontain one photoreactive group offer additional advantages,

because the photoreactive group is stable until it is exposed tohigh intensity UV light.

3. Zero-Length Cross-Linkers

The smallest reagent systems available for chemical cross-linking are zero-length cross-linkers. Those compounds mediatecross-linking between two proteins by creating a bond without an

intervening linker. Carbodiimides are probably the most widelyused type of zero-length cross-linkers, which are applied tomediate amide bond formation between a carboxylate and anamine group (or a phosphoramidate bond formation between a

phosphate and an amine) (Hoare & Koshland, 1966; Ghosh et al.,

FIGURE 3. Different types of cross-linking reagents. A: Homobifunc-

tional cross-linker, (B) heterobifunctional cross-linker, (C) trifunctional

cross-linker, and (D) heterobifunctional, cleavable cross-linker.

& SINZ

670


9/20

1990); EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)is the most popular representative (Hermanson, 1996). EDC ismostly applied in combination with sulfo-NHS (Staros, Wright,& Swingle, 1986).The purpose of adding sulfo-NHS to EDC istoincrease the stability of the active intermediate, which ultimatelyreacts with the amine group according to Scheme 1C. EDC/sulfo-

NHS-coupled reactions are highly efficient and usually increasethe yield of cross-linked product formation compared to EDCalone (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 ofthe heterobifunctional cross-linker concept with the additional

third functional group being able to specifically link to a thirdprotein or being used for affinity purification of cross-linkercontaining 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., fivetrifunctional cross-linking reagents were synthesized, includingtwo or more of the following groups: an amine-reactive NHSester, a sulfhydryl-reactive maleimide, a photochemicallyreactive benzophenone, an isotope tag, a biotin handle, and/or abaselabile ester cleavage site. The incorporation of isotope labels

into a biotinylated, trifunctional cross-linker is likely to becomeoneof the most promising strategies to design novel cross-linkingreagents. 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-linkerpossesses one amine-reactive and one photoreactive site, is

cleavable by reducing agents, and additionally allows anaffinity-based enrichment of cross-linker-containing species.

IV. MASS SPECTROMETRIC ANALYSIS

OF CROSS-LINKED PRODUCTS

The main challenges to identify cross-linked products by massspectrometry 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 analyzecross-linking 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; Menzelet al., 2001; Dreisewerd et al., 2001; Karas & Kruger, 2003).MALDI generated a great demand for a mass analyzer ideallysuited to be used in conjunction with a pulsed ion source, such asthe time-of-flight (TOF) analyzer. The performance of TOFinstruments has increased tremendously during the past fewyears. 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

products. By conducting MS/MS experiments of cross-linked

peptides, sequence information of the cross-linked peptidesand information on the sites of cross-linking both becomeavailable. MALDI-TOF-MS has been applied in numerousstudies to analyze cross-linking reaction mixtures; for exampleas 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), Muller et al. (2001);Pearson, Pannell, & Fales (2002), Sinz & Wang (2001); Backet al. (2002a,b), DAmbrosio 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 onereport, MALDI-quadrupole ion trap (QIT) mass spectrometryhas 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 electricfield, forming small, highly charged droplets. ESI requires a

sample that is devoid on non-volatile salts and detergents toobtain the highest sensitivity as well as careful optimization ofelectrospray conditions for the specific compound underinvestigation. Miniaturization of the electrospray technique(nano-electrospray), by applying narrower spray capillaries,

results in smaller droplets, reduced flow rates, and improvedsensitivity (Wilm & Mann, 1994, 1996). The peptide mixture isusually introduced into the mass spectrometer by a separationtechnique such as liquid chromatography (LC) or capillaryelectrophoresis (CE). Complex peptide mixtures are mostly

separated by reversed-phase high performance liquid chromato-

SCHEME 2. Chemical structure of the trifunctional cross-linker sulfo-

SBED.


671


10/20

graphy (RP-HPLC). ESI-MS/MS is the method of choice toobtain further information on the amino acid sequence of cross-linked peptides as well as on the cross-linked amino acids. MSexperiments using ESI-QIT, ESI-QqTOF, or ESI-TOF instru-ments are frequently used for a detailed analysis of cross-linkedproducts (Chen, Chen, & Anderson, 1999; Back et al., 2001,

2002a,b; Egnaczyk et al., 2001; Muller 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; Fuzesi et al., 2005;Onisko et al., 2005; Silva et al., 2005).

C. Bottom-Up and Top-Down

Analysis by ESI-FTICR-MS

Fora confident assignment of cross-linker containing species, theapplication of high resolution and high mass accuracy methods,such as FTICR mass spectrometry, is a valuable prerequisite

(Comisarov & Marshall, 1974; Marshall, 2000). With its ultra-high resolution and mass accuracy FTICR-MS offers thepossibility of unambiguously identifying cross-linked speciesbased 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 toanalyze cross-linked products with similar mass accuracy.

In FTICR mass spectrometers, precursor-ion selection isaccomplished by storing the ions of interest, whereas all others

are ejected by means of a suitably tailored excitation pulse; forexample, using the SWIFT technique (Guan & Marshall, 1996).MS/MS experiments are performed, using SORI-CID (Gauthier,Trautman, & Jacobson, 1991), IRMPD (Little et al., 1994), orECD (Zubarev, Kelleher, & McLafferty, 1998; Zubarev, 2003).

Alternatively, with FTICR mass spectrometers that possess aquadrupole ora linearion trapin front ofthe ICR cell it ispossibleto conduct MS/MS experiments prior to the ICR cell (Gershon,2003). ESI-FTICR mass spectrometry has been coupled on-linewith capillary and nano-liquid chromatography for high-throughput peptide identification with high sensitivity (Shenet al., 2001; Ihling et al., 2003). In our group, we have been usingnano-HPLC/nano-ESI-FTICR-MS to analyze cross-linking mix-tures 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 (Schulzet al., 2004; Kalkhof et al., 2005; Schmidt et al., 2005; Sinz,

Kalkhof, & Ihling, 2005). Using ambiguous distance restraintsderived from the chemical cross-linking datain combination with

recently developed computational methods of conjoined rigidbody/torsion angle-simulated annealing, we were able togenerate low-resolution three-dimensional structure models ofthe calmodulinmelittin complex (Fig. 4), for which no high-resolution structure exists to date (Schulz et al., 2004).

ESI-FTICR mass spectrometry is the method of choice fortop-down analyses (Fig. 1B) and has so far been successfullyemployed to assign cross-linked products from intra-molecularlycross-linked proteins, such as rhodopsin (Novak et al., 2005) orubiquitin. In the case of bovine rhodopsin, cyanogen bromide

fragments of the cross-linked protein were subjected to FTICR-

MS/MS experiments, using CID, IRMPD, and ECD. In ECD,reduced radical cations [M nH](n-1) . are generated uponcapture of electrons, which dissociate by fast and facilefragmentation of the NCabond of the peptide chain, producingmainly c and z. type fragment ions (Zubarev, Kelleher, &McLafferty, 1998).

When analyzing intra-molecular cross-linked products ofbovine rhodopsin, only ECD revealed full palmitoylation ofadjacent cysteines and cross-linking of a lysine residue to two

other lysines and one cysteine residue based on the presence of anumber of crucial c- and z.-type ions (Fig. 5).

V. IDENTIFICATION OF

CROSS-LINKED PRODUCTS

As mentioned above, mass spectrometric identification of cross-linked products can be hampered by the inherent complexity ofthe cross-linking reaction mixtures. In Figure 6, the ESI-FTICR

mass spectrum of a cross-linking reaction mixture is shown, inwhich theN-terminal domains (LN-domains) of lamininb1 andlamining1 (possessing molecular weights of ca. 59 and 55 kDa)have been cross-linked using the homobifunctional, amine-reactive cross-linker BS3. The signals of two peptides that havebeen modified by a partially hydrolyzed cross-linker are pre-

sented as insets, thus, illustrating the low signal intensities ofcross-linker-containing species and the complexity of the massspectra. Searching for cross-linked peptides in a digestionmixture, when a bottom-up analysis is applied, is comparable to

looking for the infamous needle in the haystack. To locate theneedles (cross-linked products), a number of strategies havebeen developed that aim to facilitate the identification of cross-linked products by introducing discriminating properties or byenriching cross-linker-containing species, using specific tags.The employed strategies utilize:

isotope-labeled cross-linkers or proteins,cross-linkers with affinity tags,fluorogenic cross-linkers, andchemically 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 pep-tides to be easily detected by their distinctive isotopic patternsafter enzymatic digestion (Muller et al., 2001; Pearson, Pannell,& Fales, 2002). We employed the homobifunctional, amine-reactive NHS esters BS3 (bis[sulfosuccinimidyl]suberate) andBS2G (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 andBS2G 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

& SINZ

672


11/20

products is introduced, because every species that contained onecross-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 wasidentified, is presented in Figure 7.

Another promising strategy using 18O-labeling of thehomobifunctional amine-reactive cross-linker BS3 (Table 1)has been reported (Collins et al., 2003). A bis-18O-labeled cross-linker was synthesized, so that proteins might be cross-linked bya defined mixture of unlabeled and 18O-labeled reagent toproduce two sets of signals for crosslinked products that are

separated by 4 amu. Peptides that are modified by partiallyhydrolyzed cross-linker are easily resolved by executing asimultaneous experiment that used unlabeled BS3 in the presenceof H2

18O, 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 cross-

linkers 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 afacilitated identification of cross-linked products involved arathercomplicated labeling procedure (Chen,Chen, & Anderson,1999). All primary amino groups in the protein were reductively

methylatedin a first reaction step, andafter enzymatic hydrolysis,the newly formedN-termini of the peptides were derivatized witha 1:1 (w/w) mixture of 2,4-dinitrofluorobenzene-d0/d3. Onlycross-linked peptides reacted twice and could be discriminated in

the mass spectra by their 1:2:1 isotope pattern from the 1:1

FIGURE 4. Parallel (A) and anti-parallel (B) modes of binding of melittin in the calmodulinmelittincomplex calculated from ambiguous distance restraints derived from the cross-linking data using conjoined

rigidbody/torsion angle-simulated annealing. The aminoacid 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.]


673


12/20

pattern of the singly labeled non-cross-linked peptides using theproperties of the chromophore for chromatographic separation.

Another strategy to visualize intermolecular cross-linked pep-tides, using isotope-labeled proteins, was termed the mixedisotope cross-linking (MIX) strategy (Taverner et al., 2002).That approach involved mixing 1:1 (w/w) 15N-labeled andunlabeled (14N) protein to form a mixture of isotope-labeled and

non-labeled proteinand wasexemplified by using homodimersofcytokine interleukin 6 (14N, 14N/15N, and 15N), which werechemically cross-linked. Intermolecular cross-linked peptideswere identified by their distinctive triplets or quadruplets in their

ESI mass spectra. In contrast, intramolecularly cross-linked ornon-cross-linked peptides appeared as doublets in the massspectra, thus offering the possibility to discriminate betweeninter- and intramolecular cross-linked species.

3. 18O-Labeling During Proteolytic Digestion

Incorporation of18O from isotopically enriched water into the C-termini of proteolytic peptides also presents a valuable means todiscriminate cross-linked products. The mass shifts rely on thecomplete incorporation of two 18O atoms during proteolytichydrolysis with trypsin for each C-terminus of a lysine-or

arginine-containing peptide. Acid- and base-catalyzed back-exchange with concomitant loss of the isotope label can occur atextreme pH values, but under the mild acidic conditions typicallyused for ESI- and MALDI-MS, 18O-containing carboxyl groups

of peptides are sufficiently stable (Schnolzer, Jedrzejewski, &

Lehmann, 1996). When using 18O labeling of water in thedigestion buffer, cross-linked peptides are readily distinguished

in the mass spectra by a characteristic shift of 8 amu due to theincorporation of two 18O atoms in each C-terminus. Non-cross-linked, intramolecularly cross-linked peptides as well as peptidesthat are modified by a partially hydrolyzed cross-linker containone N-terminus and show mass shifts of 4 amu, whereas

intermolecular cross-linked products that contain two N-terminiexhibit a shift of 8 amu compared to their non-labeled counter-parts. That interesting approach was exemplified for theidentification of the interacting domains within the heterodimeric

complex of two DNA-repair proteins (Rad18Rad6) (Back et al.,2002b) and for probing the tertiary structure of bovine serumalbumin (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 bytandem mass spectrometry.

B. Fluorescence Labeling

A selection of cross-linked products from the complex reactionmixtures can also be performed using the homobifunctional,

fluorogenic (fluorescence creating) cross-linking reagentdibromobimane (4,6-dibromomethyl-3,7-dimethyl-1,5-diazabi-cyclo[3.3.0]octa-3,6-diene-2,8-dione, DBB) (Scheme 3). Theuse of DBB in combination with MALDI-TOF-MS and ESI-MS/

MS was demonstrated to identify spatially close cysteines in the

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 DSS 6H]6 ion atm/z1140.638, composed of peptides a

(upperamino acid sequence)and b2 (lower amino acid sequence) (molecular weightof 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.

& SINZ

674


13/20

eye-lens proteing-crystallin (Sinz & Wang, 2004) and to definethe molecular interfaces between calmodulin and a C-terminalfragment of the giant muscle protein nebulin (Sinz & Wang,

2001). DBB cross-links thiol pairs that span 3 to 6 A (Kosoweret al., 1979), is non-fluorescent in solution, and becomesfluorescent when both of its alkylating groups have reacted(Kosower & Kosower, 1995). Thus, peptides that contain reactedDBB can be isolated by HPLC, using fluorescence detection

before mass spectrometric analysis. A major limitation of DBB isthat the unusual fragmentation patterns obtained in MS/MSexperiments are causedby the presence of the incorporated cross-linker (A. Sinz, personal observation).

C. Cleavable Cross-Linkers

Some cross-linkers (Fig. 3D) may be cleavedby periodate in casethey 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 mightbe cleaved during MS/MS experiments in case they containbonds that fragment during low-energy activation.

1. Chemical Cleavage

The thiol-cleavable cross-linking reagent DTSSP was applied tomap interfaces of a number of protein complexes (Bennett et al.,

2000; Back et al., 2002a; Davidson & Hilliard, 2003) and to mapthe three-dimensional structure ofa-crystallin (Peterson, Young,& Takemoto, 2004). MALDI-TOF-MS and ESI-QTOF-MS wereemployed to obtain specific peptide maps of the digested reactionmixtures before and after reduction, and the respective masses

were compared to each other. Peptides, which were not observedin the spectrum following reduction, were assigned as putativecross-links, and cross-links were confirmed on the basis ofobservation of the corresponding peptide halves of thepreviously detected cross-linked products after reduction. This

strategy, however, relies on observing the signals of cross-linking

FIGURE 6. Deconvoluted ESI-FTICR mass spectrum of the peptide mixture obtained by in-geldigestion

witha mixtureof endoproteinasesAspN andLysC from cross-linkedLN-domains of lamininb1 andlaminin

g1 (2mM ofeach protein, 200-fold excessof BS3 over protein concentration) atan incubationtimeof 15 min.

The magnified insets show the amino acid sequences 1 15 of lamining1 and 346370 of lamininb1 that

have been modified by partially hydrolyzed BS3.


675


14/20

products before and after reduction, and identification of cross-linked products might be ambiguous in case the correspondingsignals of the peptide halves are not observed after reduction.Moreover, for an assignment of the cross-linked amino acids,MS/MS experiments are needed.

2. MS/MS Cleavage

An amine-reactive, homobifunctional cross-linkerN-benzylimi-nodiacetoyloxysuccinimide (BID) was designed and applied tocross-linking of model peptides as well as for components of aprotein complex (Back et al., 2001, 2002a). A marker ion (atm/z91) was readily detected in both triple-quadrupole and QTOFtandem 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 synthesizedwith two low-energyMS/MS cleavablebonds in the spacer chain. Two RINK groups that contained amore-labile bond during low-energy activation compared topeptide bonds were incorporated into the cross-linker, usingsolid-phase chemistry (Tang et al., 2005). The new cross-linker

was used to cross-link ribonuclease S (Rnase S), a non-covalentcomplex 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 chainlength being 43 A in its fully extended conformation and in the

reagents high flexibility. Therefore, it is questionable whether

any useful structural information will be obtained with thisreagent. The presented strategy, however, seems promising andmight serve as basis to design a second generation of cross-linkers with improved properties.

D. Affinity Cross-Linkers

Affinity cross-linkers containa biotingroup in addition to the tworeactive groups of a heterobifunctional cross-linker, whichallows enrichment of cross-linker-containing species by affinitypurification on avidin beads (Alley et al., 2000). A modular

approach was developed for the synthesis of trifunctional cross-linking reagents, which were applied to a structural study of the

heterodimeric negative cofactor 2 protein complex (Trester-Zedlitz et al., 2003). Modularity guarantees a systematic

variation of the reagent, and thus allows the optimization of thereagent 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 ourgroup,we explored the

applicabilityof sulfo-SBED to mapthe interface regions betweencalmodulin and its target peptide derived from the skeletalmuscle myosin light-chain kinase (Sinz, Kalkhof, & Ihling,2005). The cross-linking reaction mixtures were subjected totryptic in-solution digestion, and biotinylated peptides (e.g.,

peptides that had been modified by the cross-linker as well as

TABLE 1. The homobifunctional, amine-reactive, isotope-labeled (d0/d4) cross-linkers

BS2G and BS3 with their respective spacer chain lengths

& SINZ

676


15/20

cross-linked peptides) were enriched on monomeric avidin beadsafter several washing step. Peptide mixtures were analyzed with

MALDI-TOF-MS and nano-HPLC/nano-ESI-FTICR-MS. Onedrawback of sulfo-SBED is that the spacer chain is much longer(23 A) than the preferred length of 8 t o 1 5 A, which isconsidered to provide the most useful distance geometryinformation 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 of9 A(Fujii et al., 2004).

VI. COMPUTER SOFTWARE FOR DATA ANALYSIS

Currently, the greatest deficits of employing chemical cross-linking and MS analysis include the lack of computer softwarethat can effectively analyze the enormous complexity of thereaction mixtures. All of the existing programs exhibit theirspecific limitations; thus, most of the cross-linked products mustbe manually assigned in the mass spectra. Intrapeptide cross-

links or peptides that have been modified by a partiallyhydrolyzed cross-linker are readily identified by performing

standard in silico-digestion procedures; for example, by usingthe ExPASy Proteomics Tool FindPept [http://www.expasy.ch].

The mass shift caused by reaction of the peptide with partiallyhydrolyzed cross-linker (e.g., in the case of NHS esters) isdefined as a modification. Cross-links between two peptides,however, are more difficult to identify. Programs that arecurrently available as Web server versions for free access

are the Automated Spectrum Assignment Program (ASAP), theMS2Assign, 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,

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) areindicated. Reprinted from Schmidt et al. (2005) with permission, copyright 2005 IM Publications.

SCHEME 3. Chemical structure of the fluorogenic cross-linker

dibromobimane (DBB).


677


16/20

Schnaible, & Hoffmann, 2001; Schnaible et al., 2002). TheCollaboratory for MS3D (C-MS3D, available at: http://ms3d.org/index.php) is a knowledge grid for scientist workingin the field of structural biology. The abbreviation MS3D (massspectrometry in three dimensions) has been created recently(Young et al., 2000) for the strategy of combining chemical

crosslinking with high-resolution mass spectrometry to gleanstructural information about proteins and other biologicalmacromolecules.

ASAPis a tool to assign MSpeptide peak lists that havebeengenerated by chemical cross-linking in a bottom-up approach.The program calculates the theoretical crosslinking possibilitiesfor a given protein sequence. Information about the cross-linker,the protease used for enzymatic digestion as well as potentialpost-translational modifications are defined by user input, and

ASAP returns putative assignments for the list of input m/zvalues. MS2Assign uses tandem MS peak lists that have beengenerated from the fragmentation of cross-linked, modified, orunmodified peptides in a bottom-up approach (Fig. 1A), whereas

MS2PRO assigns signals that originate from MS/MS spectra ofwhole proteins in a top-down approach (Fig. 1B).

SearchXLinks analyzes mass spectra of a modified, cross-linked, and digested protein provided that its amino acidsequence is known. Apart from searching for intramolecularcross-linked products, SearchXLinks can be employed to

completely characterize post-translational modifications of aprotein.

A commercially available program that contains a nicefeature to calculate crosslinked products from oneor twoproteins

is the GPMAW program (General Protein/Mass Analysis forWindows, Version 6.2.1) (http://welcome.to/gpmaw) (Peri,Steen, & Pandey, 2001). The user defines one or two proteinsequences of the protein(s) to be cross-linked as well as the cross-linking reagent that was employed (e.g., zero-length, homo-

bifunctional, or heterobifunctional cross-linker). The respectivecross-linker is defined by the amino acids that it targets, itselemental composition, and the composition of partially hydro-lyzed 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-linkingcombined with a mass spectrometric analysis of the products is

a rapidly developing area. Recent technological advances in thefield of mass spectrometry are likely to benefit the analysis of the

complex mixtures created by chemical cross-linking. Forconfidently assigning cross-linker-containing species, the appli-cation of high resolution and high mass accuracy methods is avaluable prerequisite, therefore, it is likely that FTICR massspectrometry will play a much larger role to analyze complex

biological samples in the near future. The application of ECDFTICR tandem MS for the sequence analysis of cross-linkedproteins and peptides provides high-resolution mass spectro-metric 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

to characterize cross-linked products. That strategy comprisesthe enzymatic or chemical cleavage of a high-molecular weightcross-linked protein complex into a few smaller pieces that aresubsequently subjected to MS/MS experiments using an FTICRmass 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-labeledand biotinylated (Trester-Zedlitz et al., 2003), will greatly

facilitate an unambiguous identification of cross-linked productswhen large protein assemblies are investigated.

There is still a long way ahead of us before chemical cross-linking combined with mass spectrometry will become agenerally applicable technique for rapid protein structurecharacterization. Clearly, improvements are needed and can be

expected in synthesizing novel cross-linking reagents, in betterunderstanding cross-linker reactivities, in developing innovativestrategies for an enrichment of cross-linked products orfacilitated MS detection, andprobably the most urgent task

in improving computer software for automated data analysis.

VIII. ABBREVIATIONS

BID N-benzyliminodiacetoyloxysuccinimideBSA bovine serum albuminBS2G Bis(sulfosuccinimidyl)glutarate

BS3 Bis(sulfosuccinimidyl)suberateCE capillary electrophoresisCID collision-induced dissociationDBB 4,6-dibromomethyl-3,7-dimethyl-1,5-diazabi-

cyclo[3.3.0]octa-3,6-diene-2,8-dione, Dibromo-

bimaneDSS disuccinimidyl suberateDTSSP 3,30-dithiobis(sulfosuccinimidylpropionate)DTT dithiothreitolECD electron-capture dissociation

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

ESI electrospray ionizationFTICR Fourier-transform ion-cyclotron resonance

HPLC high-performance liquid chromatographyIRMPD infrared multi-photon dissociationMALDI matrix-assisted laser desorption/ionizationNHS N-hydroxysuccinimidePICUP photoinduced cross-linking of unmodified

proteinsPIR protein-interaction reporterQIT quadrupole ion trapRP reversed phase

SDSPAGE sodium dodecyl sulfate polyacrylamide gelelectrophoresis

SORI-CID sustained off-resonance irradiation collision-induced dissociation

sulfo-SBED sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,30-dithiopropionate)

SWIFT stored-waveform inverse Fourier transformTOF time-of-flight

& SINZ

678


17/20

ACKNOWLEDGMENTS

The junior research group of A.S. is funded by the Saxon StateMinistry of Higher Education, Research and Culture and theDeutsche Forschungsgemeinschaft (DFG project Si 867/7-1).Financial support from the Thermo Electron Corporation

(Mattauch-Herzog award of the German Society for MassSpectrometry to A.S.) is also gratefully acknowledged. A.S.thanks Ms. Daniela Schulz and Mr. Stefan Kalkhof for criticallyreading the manuscript.

REFERENCES

Alley SC, Ishmael FT, Jones AD, Benkovic SJ. 2000. Mapping protein-

protein interactions in the bacteriophage T4 DNA polymeraseholoenzyme using a novel trifunctional photo-cross-linking and affinity

reagent. J Am Chem Soc 122:6126 6127.

Avrameas S, 1969. Couplingof enzymes to proteinswith glutaraldehyde. Use

of the conjugates for the detection of antigens and antibodies.Immunochemistry 6:4352.

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

12:222227.

Back JW, Artal Sanz M, de Jong L, de Koning LJ, Nijtmans LGJ, de KosterCG,Grivell LA, van derSpekH, MuijsersAO.2002a.A structureforthe

yeast prohibitin complex: Structure prediction and evidence from

chemical crosslinking and mass spectrometry. Prot Sci 11:24712478.

Back JW, Notenboom V, de Koning LJ, Muijsers AO, Sixma TK, de Koster

CG, de JongL. 2002b.Identificationof cross-linkedpeptidesfor protein

interaction studies using mass spectrometry and 18O labeling. Anal

Chem 74:44174422.

Back JW, de Jong L, Muijsers AO, de Koster CG. 2003. Chemical cross-

linking and mass spectrometry for protein structural modeling. J MolBiol 331:303313.

Bennett KL, Kussmann M, Bjork P, Godzwon M, Mikkelsen M, Srensen P,

Roepstorff P. 2000. Chemical cross-linking with thiol-cleavable

reagents combined with differential mass spectrometric peptide

mappingA novel approach to assess intermolecular protein contacts.

Protein Sci 9:15031518.

Bitan G, Teplow DB. 2004. Rapid photochemical cross-linkingA new tool

for studies of metastable, amyloidogenicprotein assemblies. Acc Chem

Res 37:357364.

Bragg PD, Hou C. 1975. Subunit composition, function, and spatial

arrangement in Ca2-activated and Mg2-activated adenosine tripho-sphatases of Escherichia coli and Salmonella typhimurium. Arch

Biochem Biophys 167:311321.

Brewer CF, Riehm JP. 1967. Evidence for possible nonspecific reactions

betweenN-ethylmaleimide and proteins. Anal Biochem 18:248.Browne DT, Kent SBH. 1975. Formationof non-amidine productsin reaction

of primary amines with imido esters. Biochem Biophys Res Commun67:126.

Brunner J. 1993. New photolabeling and cross-linking methods. Annu Rev

Biochem 62:483514.

Cai K, Itoh Y, Khorana HG. 2001. Mapping of contact sites in complex

formationbetweentransducinand light-activated rhodopsin by covalentcross-linking: Use of a photoactivatable reagent. Proc Natl Acad Sci

USA 98:48774882.

Carlsohn E, Angstrom J, Emmett MR, Marshall AG, Nilsson CL. 2004.

Chemical cross-linking of the urease complex fromHelicobacter 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 279:1530515313.

Chen X, Chen YH, Anderson VE. 1999. Protein cross-links: Universal

isolation and characterization by isotopic derivatization and electro-spray ionization mass spectrometry. Anal Biochem 273:192203.

Chu BCF, Kramer FR, Orgel LE. 1986. Synthesis of an amplifiable reporter

RNA for bioassays. Nucleic Acids Res 14:55915603.

Collins CJ, SchillingB, YoungMM, DollingerG, GuyRK. 2003. Isotopically

labeled crosslinking reagents: Resolution of mass degeneracy in the

identification of cross-linked peptides. Bioorg Med Chem Lett13:40234026.

Comisarov MB, Marshall AG. 1974. Fourier-transform ion-cyclotron

resonance spectroscopy. Chem Phys Lett 25:282283.

Cuatrecaseas P. 1972. Affinity chromatography of macromolecules. Adv

Enzymol 36:29.

Cuatrecaseas P, Parikh I. 1972. Adsorbents for affinitychromatographyUse

of N-hydroxysuccinimide esters of agarose. Biochemistry 11:2291

2299.

DAmbrosioC, TalamoF, VitaleRM, Amodeo P, Tell G, Ferrera L, Scaloni A.

2003. Probing the dimeric structure of porcine aminoacylase 1 by mass

spectrometric and modeling procedures. Biochemistry 42:44304443.

DavidsonWS, Hilliard GM. 2003. The spatial organization of apolipoprotein

A-1 on the edge of discoidal high density lipoprotein particles. J Biol

Chem 278:2719927207.

Dihazi GH, SinzA. 2003. Mapping low-resolution three-dimensional protein

structures using chemical cross-linking and Fourier transform ion-

cyclotron resonance mass spectrometry. Rapid Commun Mass

Spectrom 17:20052014.

Dorman G, Prestwich GD. 1994. Benzophenone photophores in biochem-

istry. Biochemistry 33:56615673.

Dreisewerd K, Berkenkamp S, Leisner A, Rohlfing A, Menzel C. 2001.

Fundamentals of MALDI-MS with pulsed infrared lasers. Int J Mass

Spectrom 226:189209.Egnaczyk GF, Greis KD, Stimson ER, Maggio JE. 2001. Photoaffinity cross-

linking of Alzheimers disease amyloid fibrils reveals interstrand

contact regions between assembled b-amyloid peptide subunits.

Biochemistry 40:1170611714.

Fenn JB, Mann M, Meng CK,Wong SF, Whitehouse CM. 1989.Electrospray

ionization for mass spectrometry of large biomolecules. Science 46:

6471.

Friedhoff P. 2005. Mapping proteinprotein interactions by bioinformatics

and crosslinking. Anal Bioanal Chem 381:7880.

Fujii N, Jacobsen RB, Wood NL, Schoeniger JS, Guy RK. 2004. A novelprotein crosslinking reagent for the determination of moderate

resolution protein structures by mass spectrometry (MS3-D). Bioorg

Med Chem Lett 14:427429.

Fuzesi M, Gottschalk KE, Lindzen M, Shainskaya A, Kuster B, Garty H,

Karlish SJD. 2005. Covalent cross-links between the g subunit(FXYD2) and a and b subunits of Na,K-ATPase. Biochemsitry

280:1829118301.

Gauthier JW, Trautman TR, Jacobson DB. 1991. Sustained off-resonance

irradiation for collision-activated dissociation involving Fourier trans-

form mass spectrometryCollision-activated dissociation techniquethat emulates infrared multiphoton dissociation. Anal Chim Acta

246:211225.

Gerardi RD, Barnett NW, Lewis SW. 1999. Analytical applications of

Tris(2,20-bipyridyl)ruthenium(II) as a chemiluminescent reagent. Anal

Chim Acta 378:143.

Gershon D. 2003. Proteomics technologies: Probing the proteome. Nature

424:581587.


679


18/20

Ghosh SS,Kao PM,McCue AW, ChappelleHL. 1990. Useof maleimide-thiol

coupling chemistry for efficient syntheses of oligonucleotide-enzymeconjugate hybridization probes. Bioconj Chem 1:7176.

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 proteinprotein interactions between MutL and MutHby cross-linking. J Biol Chem 279:4933849345.

Gorin G, Matic PA, Doughty G. 1966. Kinetics of reaction of N-

ethylmaleimide 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 molecularrulers. Protein Sci 10:12931304.

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 157158:537.

Hartmann FC, Wold F, Bifunctional reagents. 1966. Cross-linking of

pancreatic ribonuclease with a diimido ester. J Am Chem Soc 88:38903891.

Heitz JR, Anderson CD, Anderson BM. 1968. Inactivation of yeast alcoholdehydrogenase 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 spectro-

metry. J Am Soc Mass Spectrom 15:12371247.

Hunter MJ, Ludwig ML. 1962. Reaction of imidoesters with proteins andrelated small molecules. J Am Chem Soc 84:3491.

Hurst GB, Lankford TK, Kennel SJ. 2004. Mass spectrometric detection ofaffinity purified crosslinked peptides. J Am Soc Mass Spectrom

15:832839.

Ihling C, Berger K, Hofliger MM, Fuhrer D, Beck-Sickinger AG, Sinz A.

2003. Nano-high-performance liquid chromatography combined with

nano-electrospray ionization Fourier transform ion-cyclotron reso-nance mass spectrometry for proteome analysis. Rapid Commun Mass

Spectrom 17:12401246.

Itoh Y, Cai K, Khorana HG. 2001. Mapping of contact sites in complex

formationbetweenlight-activatedrhodopsinand transducin by covalent

crosslinking: Use of a chemically preactivated reagent. Proc Natl Acad

Sci USA 98:48834887.

Junge HJ, Rhee JS, Jahn O, Varoqueaux F, Spiess J, Waxham MN,

Rosenmund C, Brose N. 2004. Calmodulin and Munc13 form a Ca2

sensor/effector complex that controls short-term synaptic plasticity.

Cell 118:389401.

Kalkhof S, Ihling C, Mechtler K, Sinz A. 2005. Chemical cross-linking and

high performance Fourier transform ion cyclotron resonance massspectrometry for protein interaction analysis: Application to a

calmodulin/target peptide complex. Anal Chem 77:495503.

Karas M, Hillenkamp F. 1988. Laser desorption ionization of proteins with

molecular masses exceeding 10,000 Da. Anal Chem 60:22992301.

Karas M, Kruger R. 2003. Ion formation in MALDI: the cluster ionization

mechanism. Chem Rev 2003 103:427439.

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-resolutionmass spectrometry. J Am Chem Soc 121:806 812.

KiehmD, Ji TH.1977. Photochemicalcross-linkingof cellmembranesTest

for natural and random collisional cross-links by millisecond cross-

linking. J Biol Chem 252:85248531.

Kluger R, Alagic A. 2004. Chemical cross-linking and proteinprotein

interactionsA review with illustrative protocols. Bioorg Chem32:451472.

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 26:210217.

Kosower EM, Kosower NS. 1995. Bromobimane probes for thiols. MethodsEnzymol 251:133148.

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 76:33823386.

Kruppa GH, Schoeninger JS, Young MM. 2003. A top down approach to

protein structural studies using chemical cross-linking and Fouriertransformmass spectrometry. RapidCommunMass 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

325:759772.

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 15:16041611.

Little DP, SpeirJP, Senko MW, OConnorPB, McLaffertyFW.1994. Infraredmultiphotondissociationof large multiplychargedions forbiomolecule

sequencing. Anal Chem 66:28092815.

Liu SC, Fairbanks G, Palek J. 1977. Spontaneous, reversible protein cross-

linking in human erythrocyte membraneTemperature and pH-

dependence. Biochemistry 16:4066.

Lomant AJ, Fairbanks G. 1976. Chemical probes of extended biological

structuresSynthesis and properties of cleavable protein cross-linking

reagent dithiobis(succinimidyl-S-35 propionate). J Mol Biol 104:243

261.

Marshall AG. 2000. Milestonesin Fourier transform ion cyclotron resonance

spectrometry technique development. Int J Mass Spectrom 200:331356.

McLafferty FW, Fridriksson EK, Horn DM, Lewis MA, Zubarev RA. 1999.BiochemistryBiomolecule mass spectrometry. Science 284:1289

1290.

Melcher K. 2004. Newchemicalcrosslinkingmethods for the identificationof

transient proteinprotein interactions with multiprotein complexes.Curr Prot Pept Sci 5:287296.

Menzel C, Dreisewerd K, Berkenkamp S, Hillenkamp F. 2001. Mechanisms

of energy deposition in infrared MALDI-MS. Int J Mass Spectrom207:7396.

Muller DR, Schindler P, Towbin H, Wirth U, Voshol H, Hoving S, SteinmetzMO. 2001. Isotope-tagged cross-linking reagents. A new tool in mass

spectrometric protein interaction analysis. Anal Chem 73:19271934.

Novak P, Young MM, Schoeninger JS, Kruppa GH. 2003. A top-down

approach to protein structure studies using chemical cross-linking and

Fourier transformmass spectrometry. Eur J MassSpectrom9:623631.

Novak P, Haskins WE, Ayson MJ, Jacobsen RB, Schoeniger JS, Leavell MD,Young MM, Kruppa GH. 2005. Unambiguous assignment of intramo-

lecular chemical cross-links in modified mammalian membrane

proteins by Fourier transform-tandem mass spectrometry. Anal Chem

77:51015108.

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 cross-

linking and mass spectrometry: Evidence of the proximity of Gly90

amino termini in the PrP 2730 aggregate. Biochemistry 44:1010010109.

Partis MD, Griffiths DG, Roberts GC, Beechey RB. 1983. Cross-linking of

protein by omega-maleimido alkanoylN-hydroxysuccinimido esters. J

Prot Chem 2:263277.

& SINZ

680


19/20

Pearson KM, Pannell LK, Fales HM. 2002. Intramolecular cross-linking

experiments on cytochrome-c and ribonuclease A using an isotopemultiplet method. Rapid Commun Mass Spectrom 16:149159.

Peri S, Steen H, Pandey A. 2001. GPMAWA software tool for analyzing

proteins and peptides. Trends Biochem Sci 26:687689.

Peterson JJ, Young MM, Takemoto LJ. 2004. Probinga-crystallin structure

using chemical cross-linkers and mass spectrometry. Molecul Vision10:857866.

Petrotchenko EV, Pedersen LC, Borchers CH, Tomer KB, Negishi M. 2001.

The dimerization motif of cytosolic sulfotransferases. FEBS Lett

490:3942.

Pierce Perbio. 2003/2004. Applications handbookand catalog, Rockford, IL.

Prescher JA, Bertozzi CR.2005. Chemistry in living systems. NatChem Biol1:1321.

Rappsilber J, Siniossoglou S, Hurt EC, Mann M. 2000. A generic strategy to

analyze the spatial organization of multi-protein complexes by cross-

linking and mass spectrometry. Anal Chem 72:267275.

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.

SchmidtA, KalkhofS, Ihling C, Cooper DMF, SinzA. 2005. Mappingprotein

interfaces by chemical cross-linking and fticr mass spectrometry:

Application to a calmodulin/adenylyl cyclase 8 peptide complex. Eur JMass Spectrom 11:525534.

SchnaibleV,Wefing S, ResemannA, Suckau D, Bucker A, Wolf-Kummeth S,

HoffmannD. 2002. Screeningfor disulfidebondsin proteinsby MALDI

in-source decay and LIFT-TOF/TOF-MS. Anal Chem 74:49804988.

Schnolzer 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 deso-

rption/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 thecalmodulin-melittin complex by chemical cross-linking and high-

resolution FTICR-MS: Direct demonstration of multiple bindingmodes. Biochemistry 43:47034715.

ShenY, ZhaoR, Belov ME, ConradsTP,Anderson GA, TangK, Pasa-TolicL,

Veenstra TD, Lipton MS, Smith RD. 2001. Packed capillary reversed-

phase liquid chromatography with high-performance electrosprayionization Fourier transform ion cyclotron resonance mass spectro-

metry for proteomics. Anal Chem 73:17661775.

Silva RAGD, Hilliard GM, Fang J, Macha S, Davidson WS. 2005. A three-dimensional molecular model of lipid-free apolipoproteins A-I

determined by cross-linking/mass spectrometry and sequence thread-

ing. Biochemistry 44:27592769.

Sinz A. 2003. Chemical cross-linking and mass spectrometry for mapping

three-dimensional structures of proteins and protein complexes. J Mass

Spectrom 38:12251237.

Sinz A. 2005. Chemical cross-linking and FTICR mass spectrometry for

protein structure characterization. Anal Bioanal Chem 381:4447.Sinz A, Kalkhof S, Ihling C. 2005. Mapping protein interfaces by a

trifunctional cross-linker combined withMALDI-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 cross-

linker and mass spectrometry: Application to nebulincalmodulin

complexes. Biochemistry 40:79037913.

Sinz A, Wang K. 2004. Mapping spatial proximities of sulfhydryl groups in

proteins using a fluorogenic cross-linker and mass spectrometry. Anal

Biochem 331:2732.

Smyth DG, Konigsberg W, Blumenfeld OO. 1964. Reactions of N-ethylmaleimide with peptides and amino acids. Biochem J 91:589.

Staros JV. 1988. Membrane-impermeant cross-linking reagentsProbes of

the structure and dynamics of membrane proteins. Acc Chem Res21:435441.

Staros JV, Wright RW, Swingle DM. 1986. Enhancement by N-hydro-

xysulfosucccinimide of water-soluble cabodiimide-mediated coupling

reactions. Anal Biochem 156:220222.

Suchanek M, Radzikowska A, Thiele C. 2005. Photo-leucine and photo-methionin allow identification of proteinprotein interactions in living

cells. Nature Meth 2:261267.

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 proteinprotein inter-

actions. Anal Chem 77:311318.

Taverner T, Hall NE, OHair RAJ, Simpson RJ. 2002. Characterization of an

antagonist interleukin-6dimer by stable isotope labeling,cross-linking,

and mass spectrometry. J Biol Chem 277:4648746492.

Trakselis MA, Alley SC, Ishmael FT. 2005. Identification and mapping of

proteinprotein interactions by a combination of cross-linking,cleavage, and proteomics. Bioconjug Chem 16:741750.

Trester-Zedlitz M, Kamada K, Burley SK, Fenyo D, Chait BT, Muir TW.

2003. A modular cross-linking approach for exploring protein

interactions. J Am Chem Soc 125:24162425.

Van Dijk ADJ, Boelens R, Bonvin AMJJ. 2005. Data-driven docking for the

study of biomolecular complexes. FEBS J 272:293312.

Vasilescu J, Guo X, Kast J. 2004. Identification of proteinprotein

interactions using in vivo cross-linking and mass spectrometry.

Proteomics 4:38453854.

Wefing S, Schnaible V, Hoffmann D. 2001. SearchXLinks, http://

www.searchxlinks.de/, center of advanced european studies andresearch (caesar), Bonn, Germany.

Wilbur DS. 1992. Radiohalogenation of proteinsAn overview of radio-

nuclides, labeling methods, and reagents for conjugate labeling.

Bioconj Chem 3:433470.

WilmM, Mann M. 1994. Electrospray and Taylor-Cone theory, Doles 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 68:18.

Wine RN, Dial JM, Tomer KB, Borchers CH. 2002. Identification of

components of protein complexes using a fluorescent photo-cross-

linker and mass spectrometry. Anal Chem 74:19391945.

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 sequencingof peptidesusing MALDITOF/TOF. JAm Soc MassSpectrom13:784

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 97:58025806.

Zenobi R, Knochenmuss R. 1999. Ion formation in MALDI-MS. Mass

Spectrom Rev 17:337366.

Zubarev RA. 2003. Reactions of polypeptide ions with electrons in the

gasphase. Mass Spectrom Rev 22:5777.

Zubarev RA, Kelleher NL, McLafferty FW. 1998. Electron capturedissociation of multiply charged protein cations. A nonergodic process.

J Am Chem Soc 120:3265 3266.


681


20/20

Andrea Sinzreceived her degree in Pharmacy from the University of Tu bingen (Germany) in1993. 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 groupProtein-Ligand Interaction by Ion Cyclotron Resonance Mass Spectrometry at the

Biotechnol

Documents

Three-dimensional Protein Structures