Immunoproteomics. A. W. Purcell and J. J. Gorman. Review MASS SPECTROMETRY-BASED METHODS TO STUDY THE TARGETS OF THE IMMUNE RESPONSE*

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1 Review Immunoproteomics MASS SPECTROMETRY-BASED METHODS TO STUDY THE TARGETS OF THE IMMUNE RESPONSE* A. W. Purcell and J. J. Gorman The mammalian immune system has evolved to display fragments of protein antigens derived from microbial pathogens to immune effector cells. These fragments are typically peptides liberated from the intact antigens through distinct proteolytic mechanisms that are subsequently transported to the cell surface bound to chaperone-like receptors known as major histocompatibility complex (MHC) molecules. These complexes are then scrutinized by effector T cells that express clonally distributed T cell receptors with specificity for specific MHCpeptide complexes. In normal uninfected cells, this process of antigen processing and presentation occurs continuously, with the resultant array of self-antigen-derived peptides displayed on the surface of these cells. Changes in this peptide landscape of cells act to alert immune effector cells to changes in the intracellular environment that may be associated with infection, malignant transformation, or other abnormal cellular processes, resulting in a cascade of events that result in their elimination. Because peptides play such a crucial role in informing the immune system of infection with viral or microbial pathogens and the transformation of cells in malignancy, the tools of proteomics, in particular mass spectrometry, are ideally suited to study these immune responses at a molecular level. Here we review recent advances in the studies of immune responses that have utilized mass spectrometry and associated technologies, with specific examples from collaboration between our laboratories. Molecular & Cellular Proteomics 3: , The mammalian immune system has evolved to act as a sentinel for changes in the body that may be associated with pathological processes. To do this, it requires a mechanism whereby normal healthy cells are distinguished from infected or malignant cells. Early studies characterized this as the self-nonself paradigm, and distinction between these two states was broadly defined as the function of the immune system. This model was challenged more recently where it was suggested that the immune system functioned to recognize danger signals (1, 2), which, for example, may be in the From the Department of Microbiology and Immunology and ImmunoID, The University of Melbourne, Victoria 3010, Australia; and Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia Received, November 27, 2003 Published, MCP Papers in Press, January 8, 2004, DOI /mcp.R MCP200 form of bacterial cell wall components, viral nucleic acids, or uncontrolled malignant growth. Thus in most cases, immune responses are initiated following activation of immune effector cells through the presence of danger signals and specific recognition of foreign peptides derived from pathogen antigens or neoantigens found in cancerous cells. The response toward pathogens in a naïve individual involves an initial nonspecific response facilitated by innate immune mechanisms followed shortly afterward via the adaptive immune response. Two arms of adaptive immunity, the antibody or humoral immune response and the cellular immune response, act in concert to combat infection and malignancy in an antigenspecific manner. Fig. 1 depicts the different arms of the adaptive immune response and highlights both the cell types and MHC molecules involved in the recognition process. The first arm of the cellular immune response includes T helper lymphocytes that control antibody-secreting cells and the generation of cytotoxic T lymphocytes, the second major effector arm of the adaptive immune system. Antigen-presenting cells (APC) 1 sample both endogenous and exogenous proteins and display the proteolytically excised peptide antigens generated through antigen processing to the effector cells and molecules of the immune system, in a process that is analogous to a complex cellular proteomics experiment. However, the recognition of peptide antigens by T lymphocytes and by antibodies is exquisitely specific and sensitive, and in most cases the sensitivity of this functional recognition in vivo greatly exceeds that of modern proteomics procedures. Nevertheless, delineation of many of the molecular events involved in antigen processing has been facilitated by modern proteomics techniques. This review will describe some of the molecular events involved in antigen processing by the two arms of the cellular immune response, how proteomics has impacted on our current understanding of these processes and the potential for the latest technologies to enhance our knowledge. 1 The abbreviations used are: APC, antigen-presenting cell; MHC, major histocompatibility complex; CTL, cytotoxic T lymphocyte; ER, endoplasmic reticulum; TAP, transporter associated with antigen processing; PLC, peptide-loading complex; DC, dendritic cell; Ii, invariant chain; CLIP, class II-associated Ii peptide; MIIC, MHC class II compartment; RP-HPLC, reverse-phase high-pressure liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; LC, liquid chromatography; MS/MS, tandem mass spectroscopy; ESI, electrospray ionization; PSD, post-source decay by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics This paper is available on line at

2 FIG. 1. The interplay of APCs, helper T lymphocytes, B cells, and CD8 T cells in the induction of humoral and cell-mediated immunity. The first step in the generation of an antibody response is the uptake of antigen by an APC. Complex antigens undergo proteolysis to form peptides, some of which are bound by MHC class II molecules and are then transported to the surface of the APC. Helper T cells that bear receptors capable of interacting with the peptide-class II complexes can then bind to the APC. Additional interactions occur through costimulatory molecules and their ligands expressed on APC and T cells, respectively. These recognition events result in the transmission of activation signals to the T cell. The activated T cell is now poised to respond to those B cells that display the same peptide-class II complexes on their surfaces, acquired as a result of internalization of the immunogen through specific surface immunoglobulin receptors (B cell receptor, BCR). It is this interaction between T cells and B cells that is termed help and results in triggering of the B cell to differentiate into a plasma cell capable of secreting antibody of the same specificity as that of the immunoglobulin receptor. The interaction of activated helper T cells with certain subsets of APC can license these APC to stimulate naïve CD8 T cells. Presentation of appropriate peptide epitopes to a naïve CD8 T cells by such an activated APC results in the generation of CD8 cytotoxic T cells that are able to recognize and kill target cells that display a viral or tumor peptide in the context of MHC class I molecules. Cytokines are also produced by each cell type, which profoundly influences the type of immune response that is elicited. Cytotoxic T Lymphocytes: The Assassins of the Immune System The cytotoxic T cell response is implemented by lymphocytes expressing the CD8 coreceptor. This coreceptor facilitates recognition of major histocompatibility complex (MHC) class I molecules complexed to antigenic peptides that are expressed on the surface of all nucleated cells. It is through these cytotoxic T lymphocyte (CTL) responses that virally infected cells, tumor cells, and sometimes even normal healthy cells are destroyed, clearing the virus or eradicating tumor cells from the host. In the case of normal tissue destruction, the result is overt autoimmune disease such as that observed in the destruction of pancreatic cells in type 1 diabetes. The structure of class I MHC molecules is well defined and consists of a polymorphic heavy chain, a monomorphic light chain ( 2 microglobulin), and an antigenic peptide (Fig. 2A). The class I heavy chain has three extracellular domains ( 1, 2 domains that together form the antigenbinding cleft, and the membrane-proximal 3 domain, which is linked to a transmembrane domain and a short cytoplasmic tail). The antigen-binding cleft is composed of an eightstranded anti-parallel -pleated sheet floor bounded by helices from the 1 and 2 domains. This peptide-binding groove accommodates an antigenic peptide typically 8 11 amino acid residues in length. Heavy chain residues that line the binding groove are the focus for the majority of class I MHC polymorphisms, which in turn determine the antigen specificity of different allelic forms of MHC molecules via the formation of several conserved depressions or pockets (denoted A F) that vary in composition and stereochemistry. The A and F pockets are located at either end of the cleft and contain conserved residues involved in hydrogen-bonding interactions with the N and C termini of the bound peptide, respectively. These interactions effectively close off each end of the cleft encapsulating the termini of the bound peptide. The A pocket is frequently shallow, while the stereochemistry of the F pocket varies significantly and contributes both to conserved interactions with the C termini as well as to the specificity of the last amino acid residue of the bound peptide ligand. The B, C, D, and E pockets contribute to the specificity 194 Molecular & Cellular Proteomics 3.3

3 FIG. 2. MHC class I molecular structure and antigen-processing pathway. A, The three-dimensional structure of a class I molecule. The class I heterodimer acts as a platform to which peptide antigen binds (shown in the green space-filling model). The class I heavy chain (shown in blue ribbon form) has three domains. 1 and 2 form the peptide-binding groove; this groove is lined with the highest density of polymorphic residues that impact on ligand specificity of the MHC molecule. The 3 domain is the membrane-proximal domain and lies adjacent to the monomorphic 2 -microglobulin molecule (shown in red ribbon form). This figure was generated from the Protein Data Bank coordinates (accession number 1N2R) of HLA B*4403 bound to an endogenous peptide derived from HLA DP chain (66, 187). B, Antigen processing in the MHC class I pathway. 1, Protein antigen is degraded in the cytoplasm through the actions of the proteasome a multi-subunit protease complex with several defined proteolytic activities, some of which are induced through proinflammatory cytokines. 2, Peptides generated by the proteasome and by other cytosolic proteases are transported into the lumen of the ER in an ATP-dependent manner through the actions of the TAP heterodimer. 3, Nascent class I heavy chain is targeted to the ER and stabilized by interacting with the chaperone calnexin. Once 2 -microglobulin associates with the class I heavy chain, calnexin is exchanged for another ER-resident chaperone, calreticulin. The association of the class I heterodimer with calreticulin is also associated with the recruitment of other members of the PLC, including tapasin and ERp57. ERp57 is a thiol oxidoreductase (188, 189) involved in assuring correct disulfide bonding of the class I heavy chain ( ), and tapasin is a 48-kDa glycoprotein that bridges peptide-receptive class I heterodimers to the TAP heterodimer ( ). 4, Once a peptide of sufficient affinity binds to the class I heterodimer, this complex dissociates from the PLC and is transported to the cell surface, where it may be recognized by CD8 T cells (5). of the central portion of the bound peptide. Our understanding of binding specificity and its relationship to MHC polymorphism has come both from structural studies of class I molecules that bind to different peptide antigens and from the biochemical analysis of peptides that are bound by different class I molecules. Typically biochemical analysis involves either Edman chemistry and/or mass spectrometry (3 24). Protein antigens destined for the class I-processing pathway are degraded to oligopeptides predominantly in the cytoplasm through the action of a multi-catalytic protease structure known as the proteasome. The proteasome can exist in several different forms, depending on the exposure of the APC to proinflammatory stimuli (16, 25 41). These different forms of the proteasome engender alternate proteolytic activities and consequently produce a different array of peptide precursors for transport into the lumen of the endoplasmic reticulum (ER). Transport of these peptides occurs in an energydependent manner through a member of the ATP binding cassette transporter family known as TAP (transporter associated with antigen processing). The loading of these peptides into the binding cleft of nascent class I molecules involves a number of ER-resident chaperones (42 45) and is represented in Fig. 2B. Colocalization of a complex of the class I heavy chain- 2 -microglobulin heterodimer with the chaperones ERp57 (a thiol-oxidoreductase), calreticulin, and tapasin (which collectively form a macromolecular assembly associated with maturation of peptide-mhc complexes known as the peptide-loading complex (PLC)) with the TAP facilitates the loading of peptides into the antigen-binding cleft of the class I molecules. In addition, tapasin plays a role in ligand optimization of MHC class I molecules (46 49). Once loaded with suitable peptide cargo, the class I molecule is released Molecular & Cellular Proteomics

4 FIG. 3.MHC class II molecular structure and antigen-processing pathway. A, The three-dimensional structure of a class II molecule. The class II heterodimer acts as a binding platform to which peptide antigen binds (shown in the green space-filling model). Coordinates used to generate this figure represent the murine class II molecule I-A k complexed to a hen egg lysozyme peptide (accession number 1IAK) (196). B, Class II antigen-processing pathway. Exogenous antigen is taken up by endocytosis and degraded in the early and late endosomes. The late endosome (containing antigenic peptides) fuses with class II-rich transport vesicles (containing class II heterodimers associated with Ii) to form the MIIC compartment. In the MIIC, HLA DM catalyzes the removal of Ii-derived peptide (CLIP) from the antigen-binding cleft of the heterodimers, facilitating loading with antigenic peptides. This mature class II complex is then transported to the cell surface for scrutiny by CD4 T helper cells. from the ER, traverses the Golgi network, and is ultimately transported to the cell surface, where the complex is scrutinized by CD8 T cells. The Humoral Response: T Cell-B Cell Collaboration Leads to Secretion of Antibodies In conjunction with a robust cytotoxic response, the generation of an antibody response is essential to the clearance of many pathogens. Like the T cell receptors expressed on the surface of T lymphocytes, antibodies recognize and bind to relatively short peptide sequences in the context of the intact antigen. As such they frequently display conformational dependence for binding, because they recognize molecular surfaces and not extended antigen fragments bound to MHC molecules. Antibody production is controlled by the interaction between CD4 T helper cells and B cells that express rearranged antigen-specific immunoglobulin on their cell surface. T helper cells recognize MHC class II molecules that are expressed constitutively on specialized APC such as B cells, dendritic cells (DC), and macrophages; however, initial activation of the T helper cells usually occurs through recognition of class II molecules expressed on activated DCs as depicted in Fig. 1. Class II MHC molecules acquire antigen via a pathway that is mechanistically and physically distinct to that of class I MHC molecules. Class II molecules are composed of two polymorphic polypeptide chains ( and ) forming an heterodimer, which like class I molecules combine to form the binding cleft that accommodates peptide antigen (see Fig. 3A). Class II and chains are inserted cotranslationally into the lumen of the ER where they associate to form nascent heterodimers (50). These class II heterodimers are unstable in the absence of bound peptide and are stabilized through association with a chaperone known as invariant chain (Ii). This chaperone facilitates the formation of a multimeric structure consisting of three heterodimers each associated with an Ii molecule (i.e. (Ii B) 3 ) and occludes the peptide-binding cleft, thereby preventing premature binding 196 Molecular & Cellular Proteomics 3.3

5 of endogenous ER-resident peptides to class II molecules. The Ii is also important in trafficking nascent class II molecules to the endocytic route by virtue of a N-terminal sorting signal (51). For antigenic peptides to be able to bind to class II molecules that are resident in the endocytic compartment, Ii must first be degraded to allow access to the antigen-binding cleft. Cathepsin S mediates the proteolysis of the Ii-MHC complex (52), leaving a portion of the Ii (residues ) bound to class II heterodimers. These class II-associated Ii peptides (known as CLIP) demonstrate promiscuous binding to MHC class II alleles and occlude the peptide-binding cleft of these molecules (53, 54). In order to displace CLIP from the class II binding site, antigenic peptides must have a higher binding affinity than CLIP. This peptide exchange is catalyzed by another MHC-encoded gene product HLA DM (54, 55). Antigenic peptides are loaded in specialized intracellular compartments designated MIIC (MHC class II compartments) that arise from the fusion of late endosomes with class II-rich vesicles. These compartments are rich in cathepsins, and this intersection of the endosomal and class II-processing pathways promotes loading of class II molecules with exogenous antigen fragments. The observation that core peptide epitopes exhibit ragged N and C termini by biochemical analysis of MHC class II-bound peptides suggests that N- and C-terminal exopeptidase activities further trim the peptides bound to class II molecules during their transit from MIIC to the cell surface (56). In B cells and Fc receptor-positive cells, uptake of antigen is facilitated by a receptor-mediated endocytosis that enhances processing of specific antigen or immune complexes and subsequent presentation of CD4 T cell epitopes. Receptor-mediated uptake of antigen by B cells results in the provision of T help and differentiation of the B cells into plasmaor antibody-secreting cells (as depicted in Fig. 1). The class II-processing pathway is summarized in Fig. 3B. The mode of binding and repertoire of peptide ligands bound by MHC class II molecules has also been analyzed by biochemical methods and x-ray crystallographic studies (3, 57 61) and differs in several ways from the binding of peptides to class I molecules. Peptides that bind to MHC class II molecules are typically longer than class I ligands and tend to average around 13 amino acids in length but can be considerably longer. The interactions that close the peptide-binding cleft of class I molecules are not apparent in class II molecules, allowing the termini of the bound class II peptide to project out of the ends of the cleft. The bound peptide is retained in the cleft of class II molecules by interactions between the side chains of the peptide ligand, the specificity determining pockets of the class II molecule, and a conserved hydrogen-bonding network between the peptide backbone and conserved amino acids of the antigen-binding cleft. Like class I molecules, polymorphic amino acid residues also line the pockets of the binding cleft, and both structural and biochemical studies indicate that amino acid side chains at residues 1, 4, 6, and 9 of the class II-bound peptide typically interact with these pockets, conferring allelic specificity (3) and anchoring the peptide into the cleft. It has also been suggested that the binding of ligands to MHC class II molecules is more promiscuous than the binding of peptides to MHC class I molecules due to their free termini and ability to shift binding registers, making it more difficult to define anchor residues and to predict which peptides will be able to bind particular MHC class II molecules. Thus in summary class I and class II molecules have evolved to display fragments of either endogenous and exogenous antigens to CD8 and CD4 T cells, respectively. Each antigen-loading pathway is distinct but share several common features including the use of specialized chaperones to facilitate peptide loading, intracellular trafficking, and quality control of the process. Polymorphism within both class I and class II MHC molecules generates alternate arrays of peptide ligands and alternate targets for T cell-mediated immune responses. ROLE OF PROTEOMICS IN STUDYING THE TARGETS OF T CELL IMMUNITY Defining and Refining MHC-binding Motifs and Their Use in Bioinformatic Screening Approaches for T Cell Epitope Identification The biochemical analysis of peptides isolated from mature class I or class II molecules (62) has led to an appreciation of allelic polymorphism and its influence on ligand specificity. These and allied studies have been instrumental in establishing consensus-binding motifs for MHC class I and class II molecules, and listings of certain motifs are conveniently web based (see for example Refs. 63 and 64). These motifs describe the amino acids located at critical positions along the sequence of the antigenic peptide that are responsible for making highly conserved and energetically important contacts with pockets in the binding cleft of the class I and class II molecules. These conserved residues are therefore frequently described as anchor residues. The conserved length of class I ligands makes analysis of pooled ligands amenable to Edman analysis. Known also as pool Edman sequencing, these experiments utilize Edman chemistry to identify conserved residues at defined positions of the bound ligands (65). Where particular amino acids are favored in the sequences of the bound ligands, a significant increase in the signal observed for the given anchor amino acid is found in the corresponding cycle of Edman chemistry. Both dominant anchor residues (i.e. where the majority of MHC-bound peptides share a conserved amino acid(s) at a distinct position in the ligand) and preferred or nonpreferred residues can be delineated using this technique as inferred by the abundance or lack of signal for particular amino acids in the various cycles of the Edman analysis (62). This form of analysis is less amenable to the study of class II-bound ligands because of the greater length heterogeneity of their ligands. Although pool sequencing is an excellent tool for assessing major Molecular & Cellular Proteomics

6 changes in peptide specificity for different MHC alleles, it fails to distinguish between very closely related alleles that may have substantial overlap in bound peptide repertoire. For example, we have recently demonstrated that two HLA B44 alleles share up to 95% of their ligands and only high-resolution peptide mapping studies using mass spectrometry were able to reveal these subtle but functionally important differences in ligand repertoire (66). MHC binding motifs have been used to successfully predict T cell epitopes; however, this approach is successful in de novo prediction of T cell epitopes in only 50 70% of cases even for well-studied and abundant MHC alleles. Moreover, there are numerous examples of atypical ligands possessing non-motif-based sequences, post-translationally modified ligands, or of the failure of antigen processing to liberate the candidate peptides that restrict predictive algorithms to a subset of T cell epitopes (67 76). Furthermore, many T cell responses are focused on one or two immunodominant peptides selected from the numerous potential MHC-ligands encoded within the pathogen genome (77). The participation of so few epitopes limit predictive studies because markers of immunogenicity must take into account not just peptide binding characteristics but also the abundance and density of antigen present on the cell surface, the time of expression of the antigen during the infection or pathological process, correct processing and luminal transport of the epitope, as well as the available T cell repertoire in the host organism. Nonetheless, epitope prediction remains a popular first screening method to identify candidate T cell determinants for subsequent biological validation (78 89), and predictive algorithms are frequently combined with in vitro MHC-binding assays to confirm experimentally that the predicted ligands bind to the targeted MHC molecule (80, 90). A more comprehensive approach that allows assessment of natural processing and presentation of candidate epitopes involves the direct biochemical analysis of class I or class II ligands, which has been coined as the immunoproteome (82, 83, 91 99). Discovery of T Cell Epitopes of Relevance to Anti-viral and Anti-tumor Immunity, Transplantation and Autoimmune Disease FIG. 4. Schematic representation of processes for isolating MHC-bound peptides. A variety of approaches can be taken to examine peptides bound to MHC molecules, with several optional steps highlighted by the various flow paths of the diagram, allowing additional specificity and purification of the starting material. Several approaches have been used to isolate naturally processed and presented MHC-bound peptides directly from cells; these include analysis of peptides contained within cell lysates ( ), isolation of peptides directly from the cell surface (103, 104), and immunoaffinity purification of the MHC-peptide complexes from detergent-solubilized cell lysates (65, 105). Each approach has advantages, with the later providing the best chance of epitope identification due to the additional specificity of the immunoaffinity chromatography step and subsequent simplification of the range of cellular peptides isolated. However, each method is based upon common features and assumptions that (i) upon cell lysis, peptides bound to MHC molecules are protected from intracellular and extracellular proteolysis because they are bound to the MHC receptor, and (ii) that treatment with acid dissociates bound peptides from the MHC complexes. The relationship between each approach is represented schematically in Fig. 4, this diagram highlights the variety of approaches that can be taken, and some of them are discussed below. In the first approach, peptides are extracted from whole-cell lysates following treatment with an aqueous acid solution such as 1% trifluoroacetic acid. The presence of trifluoroacetic acid also aids in the precipitation of larger proteins, leaving a complex mixture of intracellular and extracellular peptides, a proportion of which were bound to and protected by MHC molecules. Typically these preparations are fractionated by reverse-phase high-pressure liquid chromatography (RP- HPLC) and screened with a functional assay to confirm the presence of a particular T cell epitope. These fractions can also be titrated into functional assays to allow relative quantitation of known T cell epitopes extracted from the surface of different cell types ( ). In some circumstances, the peptides are amenable to sequencing of individual components of the fractionated material by mass spectrometry. Fig. 5A demonstrates an example of this approach, where a model tumor epitope was identified in an acid eluate of a murine thymoma that expresses chicken ovalbumin as a transfected gene product and surrogate tumor antigen. In this case, the epitope was already known and T cell clones specific for the SIINFEKL determinant were available to screen RP-HPLC fractions (Fig. 5B). This experiment confirmed that the naturally processed and presented form of the SIINFEKL determinant was predominantly the minimal peptide based on T cell recognition of a RP-HPLC fraction with identical retention time as the minimal synthetic peptide. Moreover, later eluting fractions that represented precursors of this peptide were also apparent in this analysis. An alternative to the acid lysis method utilizes a nonlytic 198 Molecular & Cellular Proteomics 3.3

7 FIG. 5.Identification of model determinant SIINFEKL from a lysate of chicken ovalbumin-transfected murine thymoma. The fraction comigrating with synthetic SIINFEKL and highlighted by an arrow (A) contained T cell reactivity as determined by IL-2 secretion in a T cell hybridoma screening assay (B). In addition, some later eluting fractions also displayed activity and may have represented longer precursor peptides of the SIINFEKL epitope. approach for recovering cell-surface-associated peptides. The cells are washed in an isotonic buffer containing citrate at ph 3.3; the acidic nature of this buffer facilitates dissociation of MHC-bound peptides from the cell surface without affecting cell viability (103). The great advantage of this technique is that the same cells may be harvested daily in an iterative approach for obtaining MHC-bound material. Although the specificity of this process is somewhat better for MHC-bound material than it is from whole-cell lysates, some form of biological assay is again usually necessary to locate the peptide(s) of interest prior to attempting more definitive biochemical characterization. The use of immunoaffinity chromatography dramatically improves the specificity of the peptide extraction process by incorporating an additional, orthogonal separation step. The use of appropriate monoclonal antibodies allows isolation of a single MHC allele, and some antibodies can even select a subpopulation of MHC molecules with defined molecular or functional properties (43, 106). The use of immunoaffinity chromatography to isolate specific MHC molecules provides Molecular & Cellular Proteomics

8 FIG. 6.Schematic representation of our multi-dimensional chromatography off-line MALDI-TOF analysis of the complex immunoproteome of class I molecules (A), and a comparison of class I-bound peptides isolated from the surface of untransfected parental APC (positive spectra) and HLA B*2705 transfected APC (negative spectra) (B). This analysis was performed using a Bruker Reflex mass spectrometer (Bruker-Franzen Analytik, GMBH, Bremen, Germany) operated exclusively in the reflectron mode as described elsewhere (4, 48, 66, 187). Aliquots of each fraction (1 2 l or 1% of the fraction) were mixed with an equal volume of matrix solution ( -cyano-4- hydroxycinnamic acid (10 mg/ml) in acetonitrile-ethanol 1:1 v/v), spotted uniformly onto a target, and dried for analysis. Replicate analysis and care with sample preparation can ensure high reproducibility and confidence in the differential analysis of class I ligands. the most appropriate material for identifying individual peptide ligands restricted by a known MHC allele. It is also critical to the pool sequencing experiment and peptide repertoire studies that focus on the analysis of ligands derived from a specific MHC molecule. In all the approaches discussed, the complexity of the eluates/lysates can be reduced by using cell lines that express reduced numbers of MHC alleles. For example, homozygous cell lines express a more limited number of MHC class I or class II alleles (three loci for each class), while mutant cell lines such as C1R express very low levels of endogenous class I molecules but support high-level expression of a single transfected class I molecules (107). The simplified array of MHC molecules present on the surface of such cell lines make them very attractive for examining endogenous peptides presented by individual class I alleles under normal physiological conditions (8, ) or during infection (20, 111). Following additional RP-HPLC-based fractionation, individual species can be analyzed by mass spectrometry to examine the molecular diversity of bound peptides and also to sequence individual peptide ligands using tandem MS technologies (4, 5). Visualizing the Complex Array of Peptides Presented on the Surface of APC It has been estimated that each class I or class II allele may present as many as ,000 different peptides on the surface of APC, and only a very small proportion of these peptides (1 1,000) need to be specific for T cell recognition ( ). Given that for humans any individual may express up to six different class I (two different allotypes encoded by the HLA A, B, and C loci) and six different class II allotypes (two different allotypes encoded by the HLA DR, DQ, and DP loci), the resultant peptide landscape present on the surface of the APC may be extremely complex. Fortunately this mixture can be resolved by immunoaffinity chromatography thanks to the tremendous efforts that have gone into generating monoclonal antibodies that distinguish between the different allotypes, primarily by the tissue typing and transplantation immunologists. Thus, even though only a subset of the cellular proteome may be represented at the cell surface, because peptides derived from cellular proteins are selectively bound by MHC alleles according to their allelic binding preferences and the physical location of the antigen prior to 200 Molecular & Cellular Proteomics 3.3

9 processing, it still results in a tremendous diversity of peptide ligands. High-resolution and information-rich analytical techniques are therefore required to analyze this information, and visualizing this information has presented significant bioinformatic and technical challenges. Our approach, discussed in detail below, has been to perform multidimensional chromatography followed by off-line matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The advantages of this approach is that the molecular complexity of the material can be appreciated as a function of a chromatographic index (in our case this is usually a combination of immunoaffinity chromatography to selectively isolate the MHC allotype of interest and microbore RP-HPLC; Fig. 6A) and the majority of the fractionated material is available for functional assays or for further sequence interrogation using nano liquid chromatography (LC) tandem mass spectrometry (MS/MS) and other mass spectrometry-based technologies. Notably, other groups have performed similar analyses using both MALDI and electrospray ionization (ESI) mass spectrometry of fractionated material (e.g. Refs. 20, ) and displayed them in a variety of manners including approximating the LC-MS data to a two-dimensional gel-like format (122). In our approach, we chose to perform a systematic comparison of fractions, and for close examination individual spectra were displayed in a reflection mode (124) whereby the two equivalent spectra are superimposed on the m/z axis but displayed in opposite polarity (see Fig. 6B). In this example, the repertoire of peptides expressed by class I molecules on a human mutant APC ( (125)) both prior to (positive polarity) and subsequent to (negative polarity) transfection with HLA B*2705 were compared. Subsequent multidimensional RP-HPLC of this material allowed a detailed analysis of HLA B*2705-bound ligands, and this approach was extended to study the ligand repertoire of HLA B*2705 molecules expressed in mutant cell lines with defects in their antigenprocessing machinery, revealing novel properties of this particular class I molecule and clues to its association with the autoimmune disease ankylosing spondylitis (48). Sequencing MHC-bound Peptides The challenges associated with resolving and sequencing individual peptides from the complex mixture of MHC-bound material is not unique to immunoproteomics. This type of experiment is analogous to the shotgun proteomics-type approaches that generate complex mixtures of tryptic (or other proteolytic) fragments derived from a subset of the cellular proteome (21, 94, ). Because MHC-bound peptides frequently have varied termini and the proteolytic specificities that generate them are quite diverse, confident assignment of the peptide sequence can be difficult. Similarly, it is rare to detect peptides derived from the same protein unless the study is related to infection, for example, where target antigen sequences are known or suspected (20, 120, 121, 131). These properties of MHC-bound peptides reduce confidence in their sequence assignments by MS/MS techniques and dictate the requirement for additional screening algorithms in epitope identification strategies. For example, if the binding motif for the given allele is known, this frequently can act as a initial filter for assigning fragmentation spectra derived from immunoaffinity-purified class I MHC molecules. Fig. 7 shows the identification of a prominent peptide derived from immunoaffinity-purified HLA B*4402 molecules (binding motif XE- XXXXXXF/Y) by post-source decay (PSD) in MALDI-TOF mass spectrometry and highlights the difficulties associated with the sequence assignment of class I-bound peptides, in that many do not strictly adhere to the canonical binding motifs, lack charged termini, and lack mobile protons to facilitate good fragmentation. In this example, the presence of a proline in the sequence favored internal fragment formation, further complicating sequence assignment using automated methods. In our experience, definitive identification of peptides often requires comparison to the fragmentation fingerprint of synthetic versions of the candidate sequence as well as confirmation of identical RP-HPLC retention behavior. As proteomics instrumentation becomes more accessible to a more diverse array of researchers, so to do the demands for robust techniques for protein and epitope identification. Our work has principally involved a MALDI-TOF mass spectrometry for repertoire analysis, followed by a combination of PSD-MALDI-TOF and nanoesi-qq-tof-ms/ms. Others have used different configurations of instrumentation to achieve the same end point. The use of triple quadrupole, three-dimensional ion trap, and more recently Fourier transform mass spectrometry by the Engelhardt/Hunt and other prominent groups has clearly demonstrated the power of these other techniques for epitope identification and repertoire analysis (6, 9, 16, 18, 68, 75, 110, 114, 115, 122, ). One limitation in analysis of MHC-bound peptides is operator bias in selecting peptides from extremely complex mixtures for MS/MS. Newer technologies, such as the MALDI- TOF-TOF-MS/MS instruments will allow much-higherthroughput analyses in an automated mode. This will enable many more peptide fractions to be analyzed without operator bias and make it possible to use higher-resolution collection of HPLC fractions for analysis. The reduced peptide repertoires of narrower fractions will enhance the potential to characterize many more peptides due to the reduced likelihood of the coincidences of masses of peptides in wide-fraction cuts and a lowered tendency for suppression of ionization of peptides. The flexibility of multiple MS/MS modes of other recently developed MS analyzers involving linear ion trap should also facilitate other aspects of immunoproteomics. Identification of Epitopes Using Peptide Libraries and Mass Spectrometry Epitope extraction can be used to identify both B cell and T cell epitopes. In this approach peptide libraries, which may be restricted to the context of the primary sequence of a single Molecular & Cellular Proteomics

10 FIG. 7.PSD-MALDI-TOF MS sequencing of a HLA B*4402-restricted T cell epitope. Class I-bound peptides frequently yield incomplete b- and y-series ions, and fragmentation can be dominated by the production of internal ions (regions shaded). As such, we frequently compare the fragmentation behavior of synthetic versions of the candidate epitopes to confirm sequence assignment (positive polarity spectrum). PSD-MALDI-TOF was performed using a Bruker Reflex mass spectrometer (Bruker-Franzen Analytik, GMBH) operated exclusively in the reflectron mode as described elsewhere (197, 198). antigen or completely random, are probed with a relevant receptor (either antibody or nascent MHC molecules) to allow extraction of ligands (90, ). Mass spectrometry is subsequently used to identify the extracted components rather than deconvoluting the library into separate components or using more exhaustive library screening techniques to delineate the chemical structure of the ligand ( ). Here either the antibody is incubated with the peptide library or recombinant MHC molecules are incubated with the library. For example, we recently screened class I molecules for binding to preproinsulin peptides (90). In our approach, nascent MHC class I heavy chain and 2 -microglobulin are assembled in vitro in the presence of libraries of short (8 10 amino acid residues) peptides representing the sequence of preproinsulin. After refolding of the class I molecules within this environment, potential CTL epitopes are captured or extracted from the peptide mixture. The subsequent elution of these extracted peptides from the refolded complexes and identification of bound peptides by mass spectrometry then leads to the identification of all potential class I ligands for any antigen of known sequence. This approach does not bias the epitope search for known binding motifs and is amenable to high throughput using multiple alleles to maximize MHC class I haplotype coverage for inclusion in epitope-based subunit vaccines (166, 167). This form of epitope extraction has also been used to define class I binding motifs in combination with pool Edman sequencing (159, 168). The specificity and sensitivity of the immune response is unparalleled in biology, and the use of antibodies, for example, as diagnostic and research tools has been commonplace for many years. Proteomic analysis of the peptides and antigens involved in immune responses to pathogens and to abnormal or even normal tissues presents exciting and difficult challenges to the investigator. Underlying all these studies is the power of the immunological reagents and functional readouts that provide exquisite sensitivity unrivalled by the analytical techniques we have at hand. The use of these reagents as screening tools or as a fractionation technique will drive immunoproteomics research in the future. The use of antibodies and recombinant reagents such as MHC-tetramers and other markers of immune effector cells provide opportunities to obtain large numbers of homogeneous cells from 202 Molecular & Cellular Proteomics 3.3

11 tissues and fluids using fractionation or isolation techniques such as flow cytometry and cell sorting and laser capture microscopy. When used in combination with more typical fractionation techniques such as subcellular fractionation and affinity chromatography, the degree to which resolution of specific cells can be isolated and analyzed is improved dramatically. What are the challenges that lie ahead in immunoproteomics? Well, they are very similar to those for proteomics in general. Significant challenges in bioinformatics and searching algorithms exist for automation of MS/MS sequence assignments. Subsequent to these identification methods, the ability to display the complex array of peptides that comprise the immunoproteome and correlate data from different sources still remains a major obstacle for these studies. The issue of absolute quantitation of MHC-bound peptides using isotope-labeling techniques and so forth needs to be addressed. Finally, sensitivity and sample consumption (moving toward proteomic analysis of biopsy samples) has not fully realized its potential, and techniques such as laser capture microscopy used in tandem with MS are beginning to address these issues particularly in the identification of tumor antigens and biomarkers of malignant disease ( ). * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed. apurcell@unimelb.edu.au. REFERENCES 1. Matzinger, P. (1994) Tolerance, danger, and the extended family. Annu. Rev. Immunol. 12, Matzinger, P. (1998) An innate sense of danger. Semin. Immunol. 10, Rammensee, H. G. (1995) Chemistry of peptides associated with MHC class I and class II molecules. Curr. Opin. Immunol. 7, Purcell, A. W., and Gorman, J. J. (2001) The use of post-source decay in matrix-assisted laser desorption/ionisation mass spectrometry to delineate T cell determinants. J. Immunol. Methods 249, Bonner, P. L., Lill, J. R., Hill, S., Creaser, C. S., and Rees, R. C. (2002) Electrospray mass spectrometry for the identification of MHC class I-associated peptides expressed on cancer cells. J. Immunol. Methods 262, Hunt, D. F., Henderson, R. A., Shabanowitz, J., Sacaguchi, K., Michel, H., Sevilir, N., Cox, A. L., Appella, E., and Engelhard, V. H. (1992) Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255, de Jong, A. (1998) Contribution of mass spectrometry to contemporary immunology. Mass Spectrom. Rev. 17, Paradela, A., Garcia-Peydro, M., Vazquez, J., Rognan, D., and Lopez de Castro, J. A. (1998) The same natural ligand is involved in allorecognition of multiple HLA- B27 subtypes by a single T cell clone: role of peptide and the MHC molecule in alloreactivity. J. 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