Contributions of mass spectrometry to structural immunology

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 35, 493–503 (2000)

SPECIAL FEATURE:PERSPECTIVE

Contributions of mass spectrometry to structuralimmunology

Kevin M. Downard*Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York10461-1602, USA

Mass spectrometry has made important contributions to the field of immunology in the past decade. A varietyof mass spectrometric-based techniques have been applied to study the structures of macromolecules that playa vital role in the immune response. These include traditional molecular mass measurements to identify post-translational modifications and structural heterogeneity, mass mapping of proteolysis products, sequencingby tandem mass spectrometry and conformational analysis. Antigen–antibody and other immune complexeshave been detected by mass spectrometry, providing an avenue to study macromolecular assemblies that areimportant to immune function. By virtue of the ability of mass spectrometry based techniques to analyzecomplex biological mixtures, mass spectrometry has also been employed to identify and sequence protein epitopesimportant in both the humoral and cellular immune responses. This has been achieved through a combinationof immunoaffinity and mass spectrometric techniques, and the coupling of high-performance chromatographs tomass spectrometers. These approaches are important for the identification of pathogens and show promise forthe early diagnosis of disease associated with viral and bacterial infection and malignancy. These investigationswill enable the mechanisms associated with normal and impaired immune function to be elucidated. Massspectrometry has been utilized to characterize the structure of peptide mimics, multiple antigenic peptides andother constructs in the design of synthetic immunogens. Information derived from these studies will aid in thedevelopment of novel therapeutics and vaccines. Copyright 2000 John Wiley & Sons, Ltd.

KEYWORDS: mass spectrometry; immunology; antigen; antibody; vaccines

INTRODUCTION

At first glance, the fields of immunology and mass spec-trometry appear to share little in common. Immunologyis a discipline devoted to understanding and exploitingthe intracellular and extracellular mechanisms that pro-vide a defense against infection and disease. Mass spec-trometry, on the other hand, has more physical rootsand involves the separation and detection of chargedspecies within the confines of a vacuum system usingelectric and magnetic fields. Neither Jenner nor Thom-son, who were born over a century apart but little morethan 200 km from one another, could have foreseen aconvergence of the fields that they founded. Indeed,it has taken almost 100 years since Thomson’s earlydiscoveries for mass spectrometry to be equipped tostudy molecules that play important roles in the immuneresponse.

Particular attributes of mass spectrometry in terms ofits application to the immunological sciences include

* Correspondence to: K. M. Downard, Department of Biochemistry,Albert Einstein College of Medicine, 1300 Morris Park Avenue,Bronx, New York 10461-1602, USA.E-mail: [email protected]

the ability to (i) analyze macromolecules and their com-plexes directly, (ii) study biological mixtures such as celllysates with minimal purification, (iii) obtain structuralinformation for an individual component of a mixturewithout isolation of that constituent, (iv) obtain spectraldata within seconds, and (v) perform these analyses onlow-femtomole levels of material.

This paper describes the many and varied contribu-tions of mass spectrometry to structural immunology.These include studies of antigen and antibody structure,the analysis of immune complexes, the identification andsequencing of epitopes important in both the humoral andcellular immune responses and the conformational analy-sis of macromolecules including antigens, minibodies andpeptide mimotopes.

This paper is not intended to be an exhaustive review.It is intended to provide mass spectrometrists with anincreased understanding of structural immunology andstudies in which mass spectrometry has, or can, playan important role. Equally, it is hoped the article willinform immunologists of the advances in mass spectrom-etry and stimulate an interest in its further applicationto immunology. Future contributions will rely heavily oncollaborations between scientists of both disciplines, sincethe fields of mass spectrometry and immunology are suffi-ciently advanced so that few individuals possess a detailedunderstanding of both.

Copyright 2000 John Wiley & Sons, Ltd. Received 28 December 1999Accepted 21 January 2000

494 K. M. DOWNARD

JENNER’S TEMPLE

The field of immunology grew from observations thatthose whose survived a disease seldom suffered a second,similar illness. This led English physician Edward Jennerto immunize his patients, in a small hut on his propertythat he dubbed the Temple of Vaccinia, with cowpox toprotect them from the more virulent form of smallpox thatwas pandemic in the late 18th century. So successful werethe results that vaccination, a word derived from the Latinvaccafor cow which Jenner had used to describe his treat-ment, was adopted to treat many diseases. An understand-ing of the biology underlying these experiments, however,had to wait many more years and is still incomplete.

Although many viral- and bacterial-derived diseasesthat have caused suffering and death for centuries havenow been eradicated, or at least arrested, new ones haveappeared. The human immunodeficiency virus (HIV), avirus which cripples the immune system, is often fataland current treatments only abate the progression of thedisease associated with it. New strains of historicallyfamiliar viruses such as influenza continue to afflict theworld’s population. A growing number of pathogens havealso been identified which are capable of eluding ourbody’s natural defense mechanisms and for which nocures or treatments exist.1 It is clear that future advancesin the field of immunology will rely more heavily ona complete understanding of the structural basis of theimmune response. This in turn will enable the samebiology which affords our own natural immune protectionto be exploited in the development of new treatments,therapeutics and vaccines.

Until recently, the size of the molecules involved inimmune recognition and binding, the molecular com-plexity of extracellular and intracellular extracts, andthe low levels of the immunologically relevant com-pounds present, have collectively challenged their anal-ysis by mass spectrometry. Recent developments in massspectrometry,2 however, have overcome many of theseobstacles and this has seen the wider application of massspectrometry to studies in the immunological sciences.

A brief introduction to the immune response follows.The remaining sections describe mass spectrometric-basedapproaches and developments for the analysis of com-pounds and molecular complexes that are of criticalimportance in the immune response. The article concludeswith a discussion of areas in which mass spectrometry canforeseeably play a larger role.

OVERVIEW OF THE IMMUNE RESPONSE

Beyond the body’s external barriers to infection, theinnate and adaptive immune systems protect us frominfectious agents such as viruses and bacteria and alsofrom ourselves in autoimmune disease and malignancy.In the innate response, an organism which enters thebody encounters phagocytic and/or natural killer cells,strategically located in the liver, lungs, spleen and sinuses,as well as a series of proteins known as complement.Proteins of the complement system interact in a cascading

mechanism to attract phagocytic cells that in turn engulfand lyse the cell membrane of the invading organism.Organisms, however, are capable of altering their shapeor structure to avoid activation of the complement system.Adaptive immunity provides a second response whichprotects us from organisms that do so.

The adaptive response is a highly specific responsewhich uses antibodies both to activate the complementsystem, stimulate phagocytic cells and bind the pathogensof an invading microorganism. Antibodies are secretedfrom cells in the bone marrow, with each B-cell pro-grammed to produce a unique antibody molecule. Theseact initially as a receptor on the surface of the cell and binda foreign antigen of complementary structure. The regionof the antigen which binds to an antibody is known asan epitope or determinant. Antibodies are produced frompopulations of B-cells that can bind millions of differ-ent antigens. The production of specific antibodies (clonalselection) is triggered by an initial encounter with a for-eign antigen molecule.

While antibodies of the B-cell response (humoral immu-nity) recognize a foreign antigen in its native intact state,they usually require help from cells that develop in thethymus gland (T-cells) in order to mature into antibody-secreting cells. Many pathogens3 are also known to invadehost cells, so that the B-cell response is unable to chal-lenge them. Accordingly, cell-mediated immunity pro-vides a further level of protection against intracellularpathogens. The T-cell receptor (TCR) at the surface ofthe cell recognizes processed antigen molecules presentedto them by a group of glycoproteins called the major his-tocompatibility complex (MHC). The ability of T-cells todetect epitopic segments of a foreign antigen in the contextof self MHC class I or II molecule enables the adaptiveimmune response to eliminate pathogens and abnormalcells, but not normal cells.

ANTIGEN STRUCTURE

Most, though not all, antigens are proteins and glyco-proteins. An antigen by definition is any molecule thatbinds to an antibody. This is a reversible process and theaffinity of binding is measured in terms of an equilibriumconstant,Ka. A molecule’s capacity to bind an antibodyis defined as its antigenicity. This term should not to beconfused with immunogenicity which defines an antigen’sability to stimulate the production of antibodies againstitself. Thus an immunogen is always an antigen, but anantigen may or may not be an immunogen.

The epitopes or determinants that are important tohumoral immunity reside on the surface of the antigenmolecule. In the case of proteins, these consist of eithera contiguous series of amino acids (a continuous epitope)or residues which are only proximal in terms of the anti-gen’s three-dimensional structure (a discontinuous or con-formational epitope). Protein antigens contain numerousepitopes, each capable of being recognized by a uniqueantibody molecule with a binding domain (paratope) thatis complementary in shape and structure. Identifying pro-tein epitopes can now be achieved by mass spectrometricmeans and various approaches that have been developedare described later in this article.

Copyright 2000 John Wiley & Sons, Ltd. J. Mass Spectrom. 35, 493–503 (2000)

MASS SPECTROMETRY IN IMMUNOLOGY 495

The structural characterization of antigens by massspectrometry is accomplished in the same manner thatother macromolecules are studied. Matrix-assisted laserdesorption ionization (MALDI)4 and electrospray ioniza-tion (ESI)5,6 techniques enable large, highly polar biopoly-mers to be introduced directly into a mass spectrometer.Molecular mass measurements of intact antigens canreveal information about their size and heterogeneity.Six gag proteins isolated from the HIV were character-ized in part by mass spectrometry.7 At least two vari-ants of gag protein p24 and eight sequence variants ofp17 were identified. ESI mass spectrometry (ESIMS)of the most abundant form of p17 identified a post-translational modification at the N-terminus. Sequencingof the proteolytic products of this viral protein by tandemmass spectrometry8 also identified two forms in whichisoleucine at position 45 was replaced by valine.7

Proteolytic mass mapping in conjunction with searchesof protein databases9–11 can provide useful informationabout an antigen’s structure. This has been demonstratedin the case of the human rhinovirus, the causative agent ofthe common cold.12 These experiments identified a post-translational modification of the N-terminus of the interiorviral protein VP4. The resistance of this protein to pro-teolysis during time-course experiments in the presenceof an antiviral inhibitor was interpreted as evidence forthe interaction of the drug with the viral capsid. Prote-olytic mass mapping in conjunction with tandem massspectrometry can also be used to characterize emergingstrains of viruses. This has recently been illustrated fortype A influenza in the context of a mass spectrometricimmunoassay where the antigenic identity of the strainwas also rapidly determined.13

Bacterial pathogens have been characterized by massspectrometry. The surface components of the bacterialmembrane play a critical role in their pathogenicity.The cell surface lipooligosaccharides fromHaemophilisinfluenzae, a leading cause of bacterial meningitis in chil-dren, have been studied using a combination of massand composition analysis, methylation and tandem massspectrometry.14 The polysaccharides of most bacteriaare T-cell independent antigens and activate solely aB-cell immune response. However, some bacteria suchas mycobacterium tuberculosis have developed ways inwhich to elude extracellular degradation through the syn-thesis of an outer capsule which protects the surfacecarbohydrate molecules.15 As a result, these organismscan survive intracellularly and cell-mediated immunity isrequired to protect the host. To define the role of structuralcomponents of an antigen in the cellular immune response,deglycosylation of a complex isolated from mycobac-terium tuberculosis was characterized by ESIMS andshown to decrease its capacity to elicit cellular immuneresponsesin vivo and in vitro.16

Since humoral immunity involves the recognition of aforeign antigen in its native state, it follows that com-plete characterization of an antigen should include stud-ies of its three-dimensional structure. With the adventof the ESI technique5,6 in which molecules are trans-ferred directly from solution to the gas phase, massspectrometry now plays an important role in studies ofprotein conformation and folding. This has largely beenachieved through hydrogen/deuterium (H/D) exchangein conjunction with mass spectrometry17 in experiments

similar to those employed in nuclear magnetic resonancebased measurements. H/D exchange mass spectrometryhas been employed in part to study the refolding ofan immunoglobulin binding domain of a bacterial pep-tostreptoccocal protein.18 Time-resolved kinetic studies ofprotein folding can also be followed by ESIMS basedon changes in the relative intensities of the multiply-charged ions that appear with denaturation.19 Alternately,ion mobility measurements can be employed to examinethe gas-phase conformations of proteins.20 New approa-ches are also being developed that allow protein structuresto be probed through their reaction with radicals.21 Thisapproach results in the covalent modification of proteinswithin the confines of an electrospray ion source.

ANTIBODY STRUCTURE

Immunoglobulins are comprised of two identical heavyand two identical light chains held together by disulfidebonds and non-covalent interactions (Fig. 1). The heavychains are¾400 amino acids in length with a molecu-lar mass of¾55 kDa. Light chains are¾200 amino acidslong with a molecular mass of¾24 kDa. The variabledomains in both the light and heavy chains enable anti-bodies to bind to a diverse range of antigen structures.Based on the structure of the conserved regions of theheavy chains, immunoglobulins are divided into severalgroups; in humans there are five such groups denoted IgG,IgA, IgM, IgD and IgE. Further variation in the struc-tures of the light chains leads to isotypes denoted� and�, and antibodies are therefore classified as IgG�, IgG�,IgA�, etc. Of the various forms, IgG is of primary impor-tance in the human immune response. IgG diffuses into theextravascular regions of the body more rapidly than otherimmunoglobulins and as such is primarily responsible for

Figure 1. Schematic representation of the structure and proteol-ysis of an immunoglobulin (Ig) molecule.


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neutralizing bacterial toxins and binding microorganismsto enhance their phagocytosis.

The large size of intact immunoglobulins has tradi-tionally challenged their analysis by mass spectrometricmeans. With the development of techniques capable ofionizing very high mass species,4–6 immunoglobulinmolecules can now be introduced into a mass spectrom-eter. Still most mass analyzers have a limited mass-to-charge range (typically<5000 u), so unless thesemolecules are detected as multiply-charged ions (z > 30)they may go undetected. Time-of-flight (TOF) instru-ments have in effect an infinite mass range and haveproved most useful in these studies. Coupled to a MALDIsource, these mass analyzers are capable of analyzingintact immunoglobulin. Figure 2(a) shows the MALDI-TOF mass spectrum of a monoclonal antibody to thehemagglutinin antigen of influenza. Under the particu-lar conditions employed, a distribution of charge states

(2C through 9C) is observed that allows a more accu-rate measure of the molecular weight to be made. Therelatively low mass resolution achieved in the analy-sis of immunoglobulins is in part associated with het-erogeneity in the carbohydrate portion of the moleculesand does not solely reflect the performance of the massanalyzer.

MALDI-TOF mass spectrometry has been used toanalyze both native and conjugated IgG monoclonalantibodies.22 Drugs conjugated to monoclonal antibod-ies have been investigated as a means to deliver anti-cancer reagents to tumor sites.23 The average drug loadingwas investigated based on the difference in the molecularmass of the conjugated antibody from its native form.22

Improved mass resolution and accuracy has been achievedusing an infrared laser24 over one operating in the ultravi-olet range and IR-MALDI may offer a number of advan-tages for ionizing compounds at this molecular weight.25

Figure 2. MALDI-TOF mass spectra of a monoclonal antibody to hemagglutinin of a type A influenza strain (a) before and (b) afterreaction with a peptide comprising residues 207 225 of the antigen.13,46

Copyright 2000JohnWiley & Sons,Ltd. J. MassSpectrom. 35, 493–503 (2000)


The macroglobulin antibody IgM is a unique immuno-globulin in that it exists as a pentamer of five Ig subunitslinked by disulfide bonds. IgM shows a high bindingaffinity for antigens which contain multiple epitopes andacts as an efficient agglutinating agent. This class ofimmunoglobulin appears in the blood at the onset of infec-tion and is of particular importance in cases of bacteremia.The direct analysis of IgM by MALDI-TOF mass spec-trometry has been reported,26 with some evidence for theformation of a singly protonated ion species,27 althoughconsiderable improvements in data quality are needed forthese spectra to be analytically useful.

The surface topography of antibodies can be investi-gated by mass spectrometric means. In a manner similarto that reported for globular proteins,28 the side-chains ofamino acid residues can be chemically modified throughlimited acylation or alkylation. The reactivity of particularamino acid side-chains is determined based on the relativeion abundances of the modified and unmodified peptidesobserved in the mass spectrum of the proteolytic products.This approach can provide complementary information tothat obtained by H/D exchange mass spectrometry.29 Theselective chemical modification of amino acid side-chainshas been used to study the surface topography of a mini-body, a 61-residue protein designed to mimic theˇ-sheetdomain of the heavy chain of a mouse immunoglobulin.30

Special care must be taken in these experiments, however,to ensure that the native conformation of the antibodyis preserved under the reaction conditions. The limitedproteolysis of the same minibody has also been reportedusing a series of proteases.31 Where the native state of theantibody is retained, cleavage sites on the surface of themolecule will be more susceptible to proteolysis. How-ever, proteolysis occurs only at a limited number of sitesin the molecule and cleavage specificity is also influencedby the nature of the neighboring amino acids at those sites.Thus both protocols are better used to provide supportingevidence for a macromolecular structure rather than forpredicting a structurea priori.

MALDI-TOF mass spectrometry has also been appliedto investigate the glycosylation levels observed in immu-noglobulins. Different levels of Ig glycosylation can leadto impairment of the immune response and are associatedwith the progression of disease. In the case of diabetes,high levels of glycosylation in IgG were observed inpatients in which the disease was poorly controlled byinsulin treatment.32 Patients suffering from nephropathy(a disease of the kidney) were also reported to displaysome differences in glycosylation levels in IgA comparedwith healthy subjects.33

The multiple-charging phenomenon of the ESI tech-nique34 allows antibodies or their fragments35,36 to bestudied by mass spectrometry on mass analyzers withlimited mass ranges. Siegelet al. have shown that intactmonoclonal antibodies and their covalently modified deri-vatives can be detected by ESI-MS.24

ANTIGEN–ANTIBODY COMPLEXES

Antigen–antibody complexes are non-covalent in charac-ter. Electrostatic interactions between oppositely chargedgroups, hydrogen bonds, hydrophobic and Van der Waals

forces all contribute to the binding of an antigen toantibody. This binding is a reversible process defined byan equilibrium constantKa which measures the affinity ofassociation in units of l mol�1. Association constants of>109 l mol�1 are typical for antigens and antibodies thatinteract and these are among the strongest non-covalentinteractions known.37

The detection of an antigen–antibody complex bymass spectrometry is a more imposing task thanthe analysis of each component. Nonetheless, non-covalent protein–protein38,39 and other macromolecularcomplexes40 have been detected by both MALDI41 andESI42,43 mass spectrometry. The operating conditions forperforming these experiments may not be typical of otheranalyses as non-covalent complexes are relatively unstableand far more prone to dissociation than covalently linkedmolecules. It is also important to note that gas-phasecomplexes are free, or at least deficient, of solventmolecules and may have different structural characteristicsthan those observed in solution.

An additional complication is the size of an intactantigen–antibody complex which challenges the limits ofmass range and resolution of most analyzers. To over-come this difficulty, either the antigen or antibody can bedegraded into smaller sub-structures. Treatment of an anti-body with the protease papain results in the formation oftwo fragments that bind antigen (Fabs) and a non-bindingsegment refered to as fragment crystallizable (Fc). In con-trast, pepsin cleaves antibody at theC-terminus of thehinge region so that the interchain disulfide bonds remainintact (Fig. 1). This produces a F(ab0/2 segment and partialpFc fragment.

As popularized in x-ray crystallographic studies, Fabfragments are often more amenable to analysis than theintact antibody and enable the interaction of antibodiesto antigen to be probed. The binding of a small organichapten to an engineered antibody fragment has beendetected by ESI-MS.35,36 Haptens are small moleculeswhich by themselves are not immunogenic, yet showimmunogenic character when coupled to a larger moleculesuch as a protein. A cyclic peptide based on the paratopeof an antibody has also been shown to bind epitopicpeptides derived from two strains of the HIV-1 virus.44

Segments of protein antigens produced through prote-olysis or chemical degradation can be used to study thebinding characteristics of an intact antibody. MALDI-based experiments have begun to play a role in theseinvestigations. It has recently been shown that under cer-tain conditions antibody–peptide complexes are resilientto the preparation of the sample and the MALDI ioniza-tion event in the context of a mass spectrometric-basedimmunoassay.13,45 The direct detection of an antibody–peptide complex by MALDI mass spectrometry has alsobeen realized. Figure 2(b) shows evidence for the forma-tion of a 1 : 1 complex between a monoclonal antibody anda peptide derived from a surface antigen of the influenzavirus.46 This peptide has been shown to comprise a deter-minant of the hemagglutinin antigen recognized by theantibody.13 Other immune complexes have also been stud-ied by the MALDI process. A 1 : 1 complex of the surfaceglycoprotein gp120 and T-cell receptor CD4 that plays acentral role in HIV infection has been detected.47

The specificity of antigen–antibody interactions enablesantigens to be selectively isolated using immunoaffinity


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techniques. The use of two monoclonal antibodies directedto different epitopes of the same antigen considerablyimproves the ability to distinguish between similar ana-lytes. These techniques can be combined with mass spec-trometric analysis where the affinity of the interactionis determined based on a quantitative measure of theion signals for antigen and a mass-resolved, non-bindingvariant.48

B-CELL HUMORAL IMMUNITY: EPITOPEIDENTIFICATION

A key feature of the humoral immune response is thespecific binding of antibody to surface regions of anantigen molecule. A number of protocols have nowbeen developed which use mass spectrometry to probeantigen–antibody interactions for the purpose of identify-ing antigenic determinants or epitopes important in theB-cell response. Przybylski and co-workers used massspectrometry49,50 to identify an epitope of a comple-ment protein C3a responsible for inflammation. Plasmadesorption mass spectrometry (PDMS)51 was used toidentify the epitope following limited proteolysis of theantigen–antibody complex and dissociation of the boundpeptide. This approach is amenable to both continuousand discontinuous epitopes. It requires, however, that theantibody is immobilized before reaction and that unboundantigen is removed by washing prior to release of theepitopic peptides.

The method of Przybylski and co-workers was lateradapted to incorporate the MALDI technique. MALDIoffers significant advantages in terms of the ease ofsample preparation and affords improved ionization anddetection efficiencies in conjunction with a TOF massanalyzer. Epitopic domains were measured by MALDI-MS following incubation of an immobilized antibody witheither digested52,53 or undigested antigen.54,55 The highspecificity of monoclonal antibodies for a single antigenfrom a cell lysate has been recently demonstrated for aTy1 Gag protein expressed in yeast. MALDI and ESImass spectrometry were used to identify a linear epitopefollowing immunoaffinity purification of the protein withan antibody conjugated to a magnetic bead.56

Two more recent protocols have alleviated the need toimmobilize the antibody prior to immunobinding or pre-cipitation. In one method, digested forms of myoglobinand heart muscle troponin were treated with native mono-clonal antibodies and the non-binding peptides were sep-arated by elution through a size exclusion microcolumn.The remaining antibody complexes were isolated, washedand dissociated prior to analysis of the epitopic peptides.57

In a second approach, the antibody–peptide complex wasnot isolated from the reaction mixture and non-bindingpeptides are selectively ionized and detected in the pres-ence of the complex.13,45

This latter procedure utilizes MALDI mass spectrom-etry to identify non-binding peptides through a directcomparison of the spectra of a mixture of proteolyticpeptides before and after reaction with monoclonal anti-body (Fig. 3).13,45 Epitopic peptides are then determinedbased on the absence or diminished relative intensityof their ions in the spectrum of the antibody reaction

Figure 3. Schematic representation of a mass spectromet-ric-based immunoassay.13,45 The peptide ion denoted by anasterisk is absent from the MALDI spectrum of the antibodyreaction mixture.

mixture versusan unreactedcontrol. The approachcanbe appliedto identify either continuousor discontinuousdeterminantswhere digestionof an antigen,or mixtureof antigens,either precedesor follows its reactionwithantibody.It hasrecentlybeenappliedto surveythe anti-genicidentity of emerging strainsof the influenzavirus.13

Despite extensivevaccinationprogramsworldwide, theinfluenzavirus haseludedattemptsto eradicateor evenarrestratesof infectionduelargely to its ability to alter itsantigenicidentity. As a result,protectiveimmunity estab-lished in responseto one influenzastrain offers little tonoprotectionfrom othervariants.Themassspectrometric-basedimmunoassaydeveloped13,45 offersameansto iden-tify the antigenicityof newviral strainswith high samplethroughputandautomation.

The identification of continuousepitopesby capillaryelectrophoresiscoupledwith massspectrometryhasalsobeen reported.58 In a similar manner, signals for theepitopic peptides are observedto disappearfrom thechromatographictracefollowing injection of monoclonalantibodyinto a digestedform of the antigen.

T-CELL-MEDIA TED IMMUNITY: ANTIGENPROCESSING AND PRESENTATION

In contrast to the extracellularhumoral response,cell-mediatedimmunity involves the recognition of a pro-cessedform of an antigen. Peptidesderived from theintracelluardegradationof antigenare presentedon thecell surfacein associationwith the MHC (Fig. 4). TheMHC complex is a glycoproteindimer comprisedof an˛ subunit non-covalentlyattachedto a smaller ˇ sub-unit. If an antigen is synthesizedwithin the cell, as inthe case of viral proteins, the peptidesbind to MHC



Figure 4. Schematic representation of antigen processing and presentation in the MHC class I pathway of the cellular immune response.

classI molecules.If the antigenis derivedexogenously,as is the case for bacterial proteins, the peptidesarebound to MHC classII molecules.ClassI and II MHCmolecules share considerablesequenceand structuralhomology. In the case of classI molecules,however,the ˛ subunithasa larger polypeptidechain (¾43 kDa)linked to a ˇ2-microglobulin. X-ray crystal structuresofclassI moleculesshowthattheˇ2-microglobulinanda˛3domainof the heavychainresemblean immunoglobulin-like structure.59 Two other˛ domainsof the heavychainremote from the cellular membraneform two extendedhelicesabovea region of ˇ-pleatedsheet.This portionof the structureforms a cavity into which a short linearsegmentof the antigenis held.60 The peptidesthat bindto the cavity of MHC classI moleculesusually contain8–10 aminoacidswhile thosethat bind to MHC classIImoleculesare between12 and 24 residuesin length andpartially protrude from the binding site. The differentpathwaysthatareinvolved in antigenprocessingandpre-sentation,and the natureof the binding cavity in bothclassI andclassII MHC molecules,leadsto considerablediversity in the sequencesof bound peptides.However,commonstructuralmotifs are evident in peptideswithinanorganismsoasto enableT-cell receptorsto distinguishcells which expressforeignantigensassociatedwith viralinfection, malignancyor diseasefrom normal cells thatexpressself antigens.

In ahealthyindividual,cytotoxicT lymphocytes(CTLs)continually appraisethe proteincontentin cells to deter-minewhethertheyhavebecomeinfectedor areassociatedwith a diseasestate.Thusthe identificationof infectedordiseasealteredcells by the presenceof unique peptidemarkers(T-cell epitopes)at their surfaceis potentiallyimportantfor thediagnosisof diseaseandthedevelopmentof new immunotherapies.Peptidesexpressedby tumorcells, for instance,can be usedto treat melanomawithpeptideprimeddendriticcellsthatinducea primaryT-cellresponse.61

Naturally processedpeptidesassociatedwith classIandclassII MHC moleculesaregenerallyisolatedusingimmunoaffinity techniques.62 MHC complexesare sepa-ratedfrom acell lysateby affinity columnchromatographyand the bound peptidesare releasedfrom the complexat low pH. The extract is a complex mixture contain-ing severalthousandpeptidecomponentsmanyof whichare present in low abundance.To obtain as little as10–1000fmol of peptide,the contentsof ¾108 cells arerequired.The complexity of thesesamplesand the lowconcentrationsof peptidepresentchallengesfor eventhemost sensitiveanalytical techniques.Nonetheless,high-performanceliquid chromatography(HPLC) in combi-nation with tandemmassspectrometryhas beenshownto provide an avenuefor the analysisand sequencingofMHC-boundpeptides.

Triple-quadrupole63 andquadrupoleion traps64 aregen-erally themassanalyzersof choicein theseinvestigationsgiventheir ability to achievecloseto unit massresolution(definedat a 10%valley) acrossthemassrange,their fastscanningcharacteristicsand detectioncapabilitiesto thelow-femtomole level. Non-scanningTOF analyzersalsoafford high sensitivitiesand can be usedfor theseanal-yses.MALDI-T OF measurements,however,suffer fromdifficulties in detectingall peptidesin extremelycomplexmixtures, the requirementthat HPLC purification of thepeptide extractsbe conductedoff-line and the need topurify peptidesto a high degreein orderto effect exopep-tidasedigestionfor sequencing.65 This latter requirementis obviatedwhen post-sourcedecay(PSD) experimentsareemployedto sequencethe peptides.66

In their pioneeringwork, Hunt et al.67 werethe first toapply massspectrometrysuccessfullyto identify MHC-associatedpeptides.Peptidesbound to HLA-A2.1, oneof the most commonclassI moleculesin humans,wereanalyzedusing a combinationof microcapillary HPLCandmassspectrometry.Some200peptidesweredetectedin the extractsof immunoprecipitatesfrom ¾108 cells


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of the HLA-A2.1 cell line. Each peptide was presentat levels of between 30 and 600 fmol. Eight peptideswere sequenced by tandem mass spectrometry on a triple-quadrupole instrument and were found to share a commonstructural motif. All were nine residues in length andcontained a hydrophobic leucine or isoleucine (low-energytandem experiments do not enable these isobaric residuesto be distinguished) at position 2 and a positively chargedresidue at position 9.

The approach has since been refined and applied tostudies of antigen presentation in mutant cell lines inwhich a number of peptides were found to be dominantover normal cells68,69 consistent with a different pathwayfor antigen processing. The methodology has also beenapplied to identify peptide epitopes unique to tumor-specific and autoimmune T-cells.70,71 This is a difficulttask since antigens associated with a particular diseaseare present in a complex mixture of peptides (¾10 000in number) derived from normal cellular proteins. Inthe case of a melanoma, a melanoma-specific peptideYLEPGPVTA has been identified through tandem massspectrometric sequencing of components unique to HPLCfractions that gave a positive response in CTL assays.70

The peptide is common to two melanoma patients and isalso recognized by CTLs from three additional patients.These results suggest that the peptide is a candidate foruse in peptide-based vaccines.

A distinct advantage of tandem mass spectrometry72 isthat it allows component peptides to be sequenced withouttheir separation. On quadrupole-based instruments, low-energy (eV) collision-induced dissociation (CID) experi-ments are performed. Two fragment ion series termed b-and y-ions predominate. These fragment ions result fromthe cleavage of the peptide bond where charge is retainedin the N- or C-terminus of the peptide respectively.

The tandem mass spectrum of an epitopic peptide rec-ognized by CTLs specific to the vesicular stomatitisvirus73 is shown in Fig. 5. Mass-to-charge differencesbetween fragments of the same ion series are used toidentify the sequence of the peptide. Peptide sequencescan be obtained from tandem mass spectral (MS/MS)data by either manual or computer-aided interpretation.74

The latter approach includes the use of algorithms whichcompare the MS/MS data with hypothetical spectra pre-dicted for peptides derived from protein and nucleic aciddatabases.75

PEPTIDE MIMOTOPES AS SYNTHETICIMMUNOGENS

The use of peptides based on B- or T-cell epitopesto stimulate an immune response has been the subjectof much interest.76,77 This follows the observation thatantibodies raised to small peptide fragments can react withthe cognate sequence of the intact protein.78 However,for such peptides to evoke humoral immunity through theproduction and proliferation of antibody-secreting cells,they should adopt a stable conformation in solution.79 Ithas been found that synthetic peptides that correspondto continuous segments of a protein generally do notinduce antibodies with a high affinity for the antigen.Furthermore, many protein epitopes are discontinuous tosome extent80 such that a linear peptide correspondingto a continuous segment of an antigen is unlikely to beimmunogenic.

There has been considerable interest in designing con-formationally restrained peptides which mimic proteinepitopes for use as components of a synthetic vaccine.81

Figure 5. ESI tandem mass spectrum of the doubly protonated ion for an MHC-associated peptide of the vesicular stomatitis virus.73



The use of peptide mimotopes as immunogens offernumerous benefits on medical and economic grounds.Peptide-based vaccines are cheaply and easily producedand elicit a targeted immune response with potentiallyfewer side-effects. They would eliminate the need to iso-late virulent pathogens from the blood or serum of infectedpatients. They are also more stable than inactivated orattenuated forms of viruses.81 This latter consideration isimportant where vaccine production takes place in regionsthat are remote from the site of vaccination.

Designing peptides based on continuous epitopes issimpler than doing so for discontinuous ones. In thelatter case, it is typical for the mimic to form part ofa larger macromolecule in order to construct a stableconformational epitope. A peptide mimic to a surfaceaccessible,̨ -helical antibody-binding domain of lactatedehydrogenase (LDH) has been reported.82 Amino acidsubstitutions were made in the native epitope (˛n) toenhance the stability of the helix in the idealized form (˛i ).Peptide (̨ i), and a larger peptide that contains a repeatsequence, have been shown to be immunogenic based onthe ability of antibodies raised to the peptides to bind tothe native protein in enzyme-linked immunoassays.83

H/D exchange ESI-MS17 has been shown to be a usefultool for studying the conformational integrity of peptidemimics.84 Reverse D/H exchange can be used to eliminateproblems associated with traces of hydrogen-rich waterpresent in forward exchange experiments. The native pep-tide and mimics to LDH were labeled with deuterium atall labile sites prior to their back exchange. The idealizedform of the peptide was shown to be more conforma-tionally restrained than the native peptide based on thelevels of deuterium that remained at labile sites over time(Fig. 6).84 Furthermore, the conformational integrity ofthe idealized form was shown by D/H exchange massspectrometry to be disrupted through the substitution of asingle lysine residue with proline.84 A measure of helical

content based on level of deuterium remaining in eachpeptide was in accord with separate circular dichroism(CD) and infrared spectroscopic measurements.85 A simi-lar study employing H/D exchange ESIMS and CD spec-troscopy has been conducted for a fusion peptide andmimics to theN-terminal domain of the human immun-odeficiency virus HIV1 glycoprotein gp41.86

PEPTIDE DELIVERY: MAPS AND PROTEINCARRIERS

The delivery of epitopic peptides or mimics at sufficientdose to produce high titers of antibody is an additionalconsideration in their use as immunogens. As has beendemonstrated for the LDH system, a construct contain-ing two copies of the peptide mimic evoked an enhancedimmune response over the smaller peptide construct.83

Peptides containing multiple copies of segments of pro-tein antigens, so-called MAPS or multiple antigenic pep-tides, can thus be effective immunogens. These speciesare conveniently characterized by mass spectrometry.87

The immunogenicity of these constructs is largely depen-dent upon their ability to stimulate both a B-cell anda T-cell response. A failure to include T-cell epitopesin a tetrameric peptide derived from malarial antigenswas implicated in a poor antibody response to thisimmunogen.88 When a T-cell epitope of the antigen wascoupled to the peptide, good responses were observed inmice.89

Increasing the number of antigenic peptides, mimicsor haptens can substantially increase immunogenicity. Inone form, a synthetic vaccine might by simply comprisedof a cocktail of peptides representing both B- and T-celldeterminants. However, in order to deliver peptides inthe form of a well-characterized immunogen, they may

Figure 6. D/H exchange profiles for the native epitope (˛n) and an idealized mimic (˛i) of lactate dehydrogenase C4 determined byESI-MS.


502 K. M. DOWNARD

be attached or conjugated to protein carriers. In an idealcase, peptides that constitute B-cell epitopes or mimicsto these determinants would be attached to the proteinantigen from which they were derived. The protein carrierthen acts as a source of T-cell determinants. This ensuresthat subsequent exposure to the infectious agent will resultin specific B- and T-cell responses.

Carriers are chosen on the basis of the number of reac-tive sites, their size and solubility and their potential toconceal or even eliminate the anti-peptide response. Thecoupling of the peptide, mimic or hapten is chosen in sucha way that its structure is not perturbed or shielded by thecarrier. Bovine serum albumin and ovalbumin are com-monly used for this purpose. MALDI mass spectrometryhas been used to characterize the number and distributionof haptens and other compounds conjugated to proteincarriers.21,90 For both practical and economic reasons,immunization should also involve the minimum numberof injections using the least amount of antigen.

FUTURE PROSPECTS FOR SYNTHETICVACCINES

Despite on-going challenges, synthetic peptide-based vac-cines appear to offer considerable promise in some appli-cations. Peptides containing B- and T-cell determinants,particularly those which contain T-cell determinants thatinteract with several MHC haplotypes within and evenbetween species, appear to be effective immunogens. Amultiple antigenic peptide comprising residues 181–210of the envelope protein gp41 of type 1 human leukemiawas shown to elicit a T-cell response in humans.91 In addi-tion, a mixture of peptides derived from the sequences ofproteins associated with malaria afforded monkeys withpartial protection from the disease.92 A synthetic pro-tein SPf66 construct based on these sequences affordeda degree protection against malaria in field trials.93

DNA-based vaccines are also the subject of much inter-est. The ability to insert DNA sequences encoding forimmunogenic proteins of viruses or bacteria into foreignDNA has led to the development of DNA immunogens.DNA coding for the antigens is placed behind a suitable

promoter within a plasmid and injected directly into themuscle. In a relatively short period of time, this approachhas been tried with preparations against viral, bacterialand parasitic agents.94 The DNA is transcribed and trans-lated primarily in the cytoplasm so that all of the majorcomponents of a specific antibody and cellular immuneresponse are generated.95 Since the DNA persists afterimmunization, cytotoxic T-cells can be continually gener-ated and one of the most striking features of this approachis the longevity of the immune response. This is advan-tageous in the treatment of antigenically variable virusessuch as HIV and influenza. Although the surface antigensof these viruses vary across strains, the internal antigenswhich contain many of the T-cell determinants are con-served so that DNA encoding a nucleoprotein should offera broad level of protection.96 Details of the mode of actionof DNA, however, are still unclear97 and the widespreaduse of DNA vaccines awaits further studies of their safetyand efficacy.96 Nonetheless, clinical trials administeringDNA that encodes for HIV and influenza antigens areunder way.94 Recent improvements in the characterizationof large nucleic acids by mass spectrometry25,98 should aidin their development.

CONCLUSIONS

An improved understanding of the structural basis for theimmune response is essential for the development of newapproaches to the prevention and treatment of disease.If past contributions are any guide, mass spectrometryshould play a significant role in future studies of thestructures of immunologically important molecules andsystems, and in the elucidation of mechanisms associatedwith normal and impaired immune function. The impor-tant developments and contributions made in just the pastfew years provide sufficient impetus for these studies tobe energetically pursued.

Acknowledgements

The author is grateful to Janna Kiselar for acquiring all of the massspectra presented in this paper and Stan Nathenson for reviewing themanuscript.

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Contributions of mass spectrometry to structural immunology