Spliced HLA-bound peptides: a Black Swan event in immunology.

Peptides that bind to and are presented on the cell surface by human leucocyte antigen (HLA) molecules play a critical role in adaptive immunity. For a long time it was believed that all the HLA-bound peptides were generated through simple proteolysis of linear sequences of cellular proteins, and therefore are templated in the genome and proteome. However, evidence for untemplated peptide ligands of HLA molecules has accumulated during the last two decades, with a recent global analysis of HLA-bound peptides suggesting that a considerable proportion of HLA-bound peptides are potentially generated through splicing/fusion of discontinuous peptide segments from one or two distinct proteins. In this review, we will evaluate recent discoveries and debates on the contribution of spliced peptides to the HLA class I immunopeptidome, consider biochemical rules for splicing and the potential role of these spliced peptides in immune recognition.

A comprehensive analysis of peptides presented by HLA-A1.

Human leukocyte antigen (HLA)-A1 is one of the most common Caucasian HLA-A alleles. Here, we describe the comprehensive analysis of the HLA-A*01:01 ligand repertoire with the identification of 4735 naturally processed and presented peptides derived from 2477 source proteins. We found HLA-A*01:01 bound an equivalent number of ligands of 9 or 10 amino acids in length as well as being remarkably tolerant of even longer peptides. Indeed close to half of the HLA-A1 bound peptides identified ranged between 11 and 13 amino acids in length. These longer peptides contained the strong canonical motif of and acidic E/D residue at position 3 (P3) and Y at the C-terminus (CΩ), a motif that was still apparent in peptides of up to 18 amino acids in length. The identification of this large database of natural ligands will facilitate the refinement of predictive algorithms particularly with respect to longer peptide ligands.

Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients.

OBJECTIVES: We evaluated whether the measurement of serum phosphorylated neurofilament heavy chain (pNF-H) titre is likely to be a valid biomarker of axonal injury in multiple sclerosis (MS). METHODS: Serum pNF-H concentrations were measured by ELISA in cases with relapsing-remitting (RR)-MS (n=81), secondary progressive (SP) MS (n=13) and primary progressive (PP)-MS; n=6) MS; first demyelinating event (FDE; n=82); and unaffected controls (n=135). A subset of MS cases (n=45) were re-sampled on one or multiple occasions. The Multiple Sclerosis Severity Score (MSSS) and MRI measures were used to evaluate associations between serum pNF-H status, disease severity and cerebral lesion load and activity. RESULTS: We confirmed the presence of pNF-H peptides in serum by ELISA. We showed that a high serum pNF-H titre was detectable in 9% of RR-MS and FDE cases, and 38.5% of SP-MS cases. Patients with a high serum pNF-H titre had higher average MSSS scores and T2 lesion volumes than patients with a low serum pNF-H titre. Repeated sampling of a subset of MS cases showed that pNF-H levels can fluctuate over time, likely reflecting temporal dynamics of axonal injury in MS. CONCLUSIONS: A subset of FDE/MS cases was found to have a high serum pNF-H titre, and this was associated with changes in clinical outcome measures. We propose that routine measurement of serum pNF-H should be further investigated for monitoring axonal injury in MS.

A comprehensive analysis of constitutive naturally processed and presented HLA-C*04:01 (Cw4)-specific peptides.

The human B lymphoblastoid cell line C1R is widely regarded as human leukocyte antigen-A (HLA-A)/HLA-B negative and is therefore frequently exploited as a recipient cell line to study HLA class I functions. However, the normal levels of HLA-C*04:01 often hamper the investigation of introduced HLA class I allomorphs, which is particularly evident in sensitive applications such as mass spectrometry. Here we describe the comprehensive analysis of endogenous HLA-C*04:01 ligands expressed on the surface of C1R cells to (i) define a large sequence dataset of HLA-C*04:01 ligands, to (ii) refine the HLA-C*04:01 peptide-binding motif and (iii) to provide a resource that allows discrimination between peptides bound to introduced HLA class I subtypes and to the endogenous HLA-C*04:01 molecules.

The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.

Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing beta cells. While the role of CD4(+) T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4(+) T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4(+) T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6-A7). Here we describe the analysis of a CD4(+) T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4(+) T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4(+) T cell responses in human T1D.

Immunodominance hierarchies and gender bias in direct T(CD8)-cell alloreactivity.

Allogeneic solid organ transplantation often occurs across multiple donor-recipient HLA mismatches with consequent risk of allograft rejection. However, there is growing evidence that not all HLA mismatches are equivalent in their stimulation of allogeneic T cells making it important to determine which of these might be more significant as predictors of allograft rejection. To this end, we used defined antigen-presenting cell (APC) transfectants expressing single MHC-I allotypes as target cells that could discriminate the relative contribution of individual mismatched MHC-I allotypes to direct T-cell alloreactivity. We demonstrate remarkably reproducible patterns of immunodominance in reactivity across mismatched MHC-I allotypes. These patterns are HLA context-dependent, partly reflecting alloantigenic competition in responder cell responses. In strong alloresponses, we also observed an increased percentage of alloreactive T(CD8) cells in female responders, regardless of the stimulator gender, highlighting HLA-independent factors in the potency of the alloresponse. This approach provides a potential measure of specific alloreactive T cells that could be used in clinical practice for selection of donors, assessment of posttransplant outcomes, modulation of immunosuppression and detection of rejection episodes.

The molecular basis of cross-reactivity in the Australian Snake Venom Detection Kit (SVDK).

The Snake Venom Detection Kit (SVDK) is of major medical importance in Australia, yet it has never been rigorously characterised in terms of its sensitivity and specificity, especially when it comes to reports of false-negative and false-positive results. This study investigates reactions and cross-reactions of five venoms the SVDK is directed against and a number of purified toxins. Snakes showing the closest evolutionary relationships demonstrated the lowest level of cross-reactivity between groups. This was, instead, far more evident between snakes that are extraordinarily evolutionary separated. These snakes: Pseudechis australis, Acanthophis antarcticus and Notechis scutatus, in fact displayed more false-positive results. Examination of individual toxin groups showed that phospholipase A(2)s (PLA(2)s) tends to react strongly and display considerable cross-reactivity across groups while the three-finger toxins (3FTx) reacted poorly in all but the Acanthophis well. The hook effect was evident for all venoms, particularly Oxyuranus scutellatus. The results of this study show considerable variation in toxin detection, with implications in further development of venom detection, both in Australia and other countries.

Immunoproteomics: Mass spectrometry-based methods to study the targets of the immune response.

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 MHC-peptide 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.

Novel strategy for identification of candidate cytotoxic T-cell epitopes from human preproinsulin.

We describe a strategy for identifying ligands of human leukocyte antigen (HLA) class I molecules based on a peptide library-mediated in vitro assembly of recombinant class I molecules. We established a microscale class I assembly assay and used a capture ELISA to quantify the assembled HLA-peptide complexes. The identity of the bound ligands was then deduced by mass spectrometry. In this method, HLA complexes assembled in vitro in the presence of components of a mixture of peptides were immunoprecipitated and the bound peptide(s) identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This process of epitope extraction is robust and can be used with complex mixtures containing in excess of 300 candidate ligands. A library of overlapping peptides representing all potential octamers, nonamers and decamers from human preproinsulin was synthesized using unique library chemistry. Peptides from the library were used to initiate assembly of recombinant HLA-B8, HLA-B15 and HLA-A2, facilitating the identification of candidate T-cell epitopes from preproinsulin.

Association of stress proteins with autoantigens: a possible mechanism for triggering autoimmunity?

Patterns of autoantibody production are diagnostic of many autoimmune disorders; the recent observation of additional autospecificities towards stress-induced proteins may also provide insight into the mechanisms by which such responses arise. Grp78 (also known as BiP) is a target of autoaggressive B and T cell responses in our murine model of anti-Ro (SS-A) autoimmunity and also in rheumatoid arthritis. In this report we demonstrate reciprocal intermolecular spreading occurs between Ro52 and Grp78 in immunized mice, reflecting physiological association of these molecules in vivo. Moreover, we provide direct biochemical evidence that Grp78 associates with the clinically relevant autoantigen, Ro52 (SS-A). Due to the discrete compartmentalization of Ro52 (nucleocytoplasmic) and Grp78 (endoplasmic reticulum; ER) we propose that association of these molecules occurs either in apoptotic cells, where they have been demonstrated indirectly to co-localize in discrete apoptotic bodies, or in B cells themselves where both Ro52 and Grp78 are known to bind to immunoglobulin heavy chains. Tagging of molecules by association with Grp78 may facilitate receptor mediated phagocytotsis of the complex; we show evidence that exogenous Grp78 can associate with cell surface receptors on a subpopulation of murine splenocytes. Given the likelihood that Grp78 will associate with viral glycoproteins in the ER it is possible that it may become a bystander target of the spreading antiviral immune response. Thus, we propose a model whereby immunity elicited towards Grp78 leads to the selection of responses towards the Ro polypeptides and the subsequent cascade of responses observed in human disease.

The central role played by peptides in the immune response and the design of peptide-based vaccines against infectious diseases and cancer.

Vaccines are one of the most cost effective methods of improving public health thereby increasing the quality of life. Prophylactic and therapeutic treatment by vaccines can prevent infectious diseases and some cancers and could also be used in the treatment of autoimmune disorders. An appreciation of this potential has resulted in a burgeoning literature which not only describes the scientific efforts being made into designing new and improved vaccines but also drives the efforts being made by public health organizations world-wide in delivering vaccines to the community. At the forefront of technologies being applied to the design of vaccines is the use of synthetic peptides; the chemical technologies used to assemble peptides have made great strides over the last decade and assembly of hi-fidelity peptides which can be of high molecular weight, multimeric or even branched is now almost routine. Together with the advances in peptide technology our understanding of the molecular events that are necessary to induce immune responses has also made great strides. The central role that peptides play in immune recognition is now recognised and rules are emerging that are being applied to the construction of peptide-based vaccines that, in the right context, can induce humoral (antibody) and cellular (cytotoxic and helper T cell) immune responses. Synthetic peptides are exquisitely placed to answer questions about immune recognition and along the way to provide us with new and improved vaccines.

The use of post-source decay in matrix-assisted laser desorption/ionisation mass spectrometry to delineate T cell determinants.

The identification of naturally processed peptides presented by molecules of the major histocompatibility complex (MHC) has progressed significantly over the past decade. The elution of peptides from immunoaffinity purified complexes of MHC class I or class II molecules has provided highly specific biochemical information regarding the nature of endogenous peptides capable of binding to and being presented by particular MHC alleles. Whilst Edman chemistry is sufficient for the identification of abundant or homogeneous immunodominant peptides contained in samples of fractionated peptides, mass spectrometry has proved more powerful for sequencing less abundant species present in the typically heterogeneous fractions of eluted peptides. This review focuses on the characterisation of T cell determinants by matrix-assisted laser desorption/ionisation (MALDI)-time-of-flight (TOF) mass spectrometry (MS). We demonstrate, with specific examples, the utility of post-source decay in MALDI-TOF MS for the characterisation of the amino acid sequences of both native and modified T cell determinants. The potential advantages and pitfalls of this technique relative to the more commonly used forms of tandem mass spectrometry in electrospray and ion spray modes of ionisation as well as hybrid quadrupole-quadrupole-TOF instruments are discussed. We highlight the complementarity between these techniques and discuss the advantages in the combined use of both MALDI- and electrospray-based instrumentation in epitope identification strategies.

Quantitative and qualitative influences of tapasin on the class I peptide repertoire.

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta(2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

Tapasin-mediated retention and optimization of peptide ligands during the assembly of class I molecules.

The murine class I H-2Kb molecule achieves high level surface expression in tapasin-deficient 721.220 human cells. Compared with their behavior in wild-type cells, Kb molecules expressed on 721.220 cells are more receptive to exogenous peptide, undergo more rapid surface decay, and fail to form macromolecular peptide loading complexes. As a result, they are rapidly transported to the cell surface, reflecting a failure of endoplasmic reticulum retention mechanisms in the absence of loading complex formation. Despite the failure of Kb molecules to colocalize to the TAP and their rapid egress to the cell surface, Kb is still capable of presenting TAP-dependent peptides in the absence of tapasin. Furthermore, pool sequencing of peptides eluted from these molecules revealed strict conservation of their canonical H-2Kb-binding motif. There was a reduction in the total recovery of peptides associated with Kb molecules purified from the surface of tapasin-deficient cells. Comparison of the peptides bound to Kb in the presence and absence of tapasin revealed considerable overlap in peptide repertoire. These results indicate that in the absence of an interaction with tapasin, Kb molecules fail to assemble with calreticulin and TAP, yet they are still capable of acquiring a diverse array of peptides. However, a significant proportion of these peptides appear to be suboptimal, resulting in reduced cell surface stability of Kb complexes. Taken together, the findings indicate that tapasin plays an essential role in the formation of the class I loading complex, which retains class I heterodimers in the endoplasmic reticulum until optimal ligand selection is completed.

Endogenous and exogenous factors contributing to the surface expression of HLA B27 on mutant APC.

We have examined the expression of HLA B*2705 in the mutant cell line 721.220, which lacks endogenous HLA A and B alleles and expresses a defective tapasin molecule. Several peptide sensitive mAbs distinguish between HLA B*2705 expressed on the surface of 721.220 cells (B27.220) and 721.220 cells co-transfected with human tapasin (B27.220.hTsn). This differential staining defines subtle differences in the conformation of HLA B27, which most likely reflect changes in the repertoire of antigenic peptides bound to B27 in the presence and absence of wild type tapasin. HLA B27 molecules expressed on the surface of 721.220 display increased levels of "free" B27 heavy chain (HC-10 staining), an epitope that is dependent on TAP-translocated peptides. The conformation and stability of B27 molecules was examined by investigating the integrity of mAb epitopes and the half-lives of these complexes on cells cultured with and without serum. The decay of surface B27 epitopes occurred more rapidly in B27.220 and this effect was exaggerated in serum free media. Importantly, the decay of surface B27 molecules in B27.220.hTsn cells was characterized by an early increase in HC-10 staining when the cells were grown in serum free media. This decay of B27 molecules via HC-10 reactive intermediates was not observed in B27.220 cells, implying molecules on these cells may already have passed through this stage prior to surface expression. Taken together these observations indicate that tapasin has a significant contribution to the composition and stability of the B27-bound peptide repertoire.

The peptide-loading complex and ligand selection during the assembly of HLA class I molecules.

The identification and characterisation of the class I peptide loading complex has resulted in an appreciation of the co-ordinated and multifaceted nature of HLA class I assembly in the lumen of the endoplasmic reticulum. This loading complex consists of the assembling class I heterodimer in association with a number of molecular chaperones. These chaperones can be classified as generic to the folding of most glycoproteins in the endoplasmic reticulum or specific to the class I loading pathway. The functions of the various components of the loading complex in class I molecule assembly are reviewed. A critical component of the class I loading complex is the specialised chaperone tapasin. The role of tapasin in the stabilisation and retention of empty or suboptimally loaded class I molecules and the facilitation of the loading of these molecules with more appropriate ligands is discussed. As such, it is proposed that tapasin is a major determinant of peptide repertoire selection for class I-restricted presentation in normal antigen presenting cells. The potential implications in vaccine design and autoimmunity are discussed.

Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments.

The isocratic and gradient elution behaviour of beta-endorphin and glucagon, two polypeptides known to exist in amphipathic alpha-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo-acetonitrile mobile phases. The effects of changes in the volume fraction, psi, of the organic solvent modifier and temperature, T, on the magnitudes of the S and log k(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k') vs. psi using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and log k(o) values, obtained from the plots of logarithmic median capacity factor (log k) versus the median volume fraction of the organic solvent modifier (psi) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo-acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and S values, or the log k(o) and log k(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for beta-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of log k vs. 1/T (corresponding to an apparent change in the median enthalpy of association, deltaH(o)assoc, or an apparent change in the median entropy of association, deltaS(o)assoc) substantially deviated from the values obtained for the thermodynamic parameters, deltaH(o)assoc and deltaS(o)assoc, derived from the log k' vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates.

Probing the binding behavior and conformational states of globular proteins in reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used technique for the separation of proteins under low pH aquo-organic solvent gradient elution conditions, typically carried out at ambient temperatures. These conditions can however induce conformational effects with proteins as evident from changes in their biological or immunological activities. By monitoring the influence of temperature on the retention and band-broadening characteristics of proteins, the role of conformational processes in these lipophilic environments can be examined. These processes can then be interpreted in terms of a two-state model involving a native (N) and a fully unfolded species (U) or more complex folding/unfolding models. In the present study, the gradient elution RP-HPLC behavior of sperm whale myoglobin (SWMYO) and hen egg white lysozyme (HEWL) has been investigated at temperatures between 5 and 85 degrees C with n-octadecyl (C18)- and n-butyl (C4)-silica reversed-phase sorbents. The interaction of these proteins with these reversed-phase sorbents has also been examined in terms of the contributions that the heme prosthetic group of SWMYO and the disulfide bonds in HEWL make to the stabilization of the native conformation of these proteins in these hydrophobic environments. The observed interconversions of multiple peak zones of SWMYO and HEWL in the presence of C18 and C4 ligands have been subsequently analyzed in terms of the unfolding processes that these proteins can undergo at low pH and at elevated temperatures. The ability of hydrocarbonaceous ligands to trap ensemblies of partially unfolded conformational intermediates of proteins in these perturbing environments has been examined. Pseudo-first-order rate constants have been derived for these processes from analysis of the dependencies on time of the concentration of the different protein species at specified temperatures. The relationship of these processes to the conformational transitions that these proteins can undergo via molten globule-like intermediates (i.e., compact denatured states with a significant amount of residual secondary structure) in solution has also been examined. This study thus further documents an experimental strategy to assess the folding/unfolding behavior of globular proteins in the presence of hydrophobic surfaces and aquo-organic solvents, whereby the system parameters can potentially affect the preservation of native conformations, and thus the function, of the protein under these conditions.

Molecular chaperones are targets of autoimmunity in Ro(SS-A) immune mice.

We have used a murine model of experimental anti-Ro(SS-A) autoimmunity to dissect additional intermolecular interactions between the 52-kD Ro (Ro52) and 60-kD Ro (Ro60) autoantigens and molecular chaperones. Immune responses to members of the heat shock protein hsp70 and hsp90 families were measured by immunoblotting and ELISA in sera from mice immunized and boosted with purified recombinant Ro52, Ro60 and La (SS-B). All Ro52 and Ro60 immune sera immunoblotted the inducible glucose-regulated protein grp78 and hsp70 species but not constitutive hsc70 or hsp90. The kinetics of antibody production and reciprocal affinity purification experiments indicated that the grp78 and hsp70 responses were cross-reactive but distinct from immune responses to the primary Ro52 and Ro60 immunogens and the endoplasmic reticulum (ER)-resident chaperone calreticulin. No responses to molecular chaperones were detected in the La-immunized mice. Control immunizations indicated that the recruited grp78 and hsp70 responses were specific for the Ro proteins and not due to immunization with denatured protein. The rapid spreading of immunity to the inducible grp78 and hsp70 in Ro52- and Ro60-immunized mice suggests that these components may co-localize and physically associate under certain physiological conditions which may promote autoimmunization. The potential importance of the ER-resident chaperones grp78 and calreticulin is further supported by their co-localization with Ro in small apoptotic membrane blebs and the finding of a novel putative grp78 binding motif in the carboxyl-terminal region of Ro52.

RP-HPLC binding domains of proteins.

Procedures have been developed to identify the chromatographic binding domains of horse heart cytochrome c (Cyt c) and bovine growth hormone (bGH) during their interaction with reversed-phase sorbent materials. The procedure involves adsorption of the protein solute to the chromatographic sorbent, followed by proteolytic cleavage. Comparison of the proteolytic map obtained for Cyt c and bGH in free solution with the corresponding map obtained when these proteins are adsorbed to the chromatographic sorbent revealed significant differences in the digestion pattern. Following characterization of the peptides generated in both maps, the results indicated that specific regions on the surface of both Cyt c and bGH are inaccessible to tryptic cleavage when adsorbed to the hydrophobic surface of both a C-4 and a C-18 sorbent. Based on the assumption that the region of the protein surface that is in contact with the sorbent remains intact and bound to the sorbent during the digestion step, while the protein surface that is exposed to the solvent is accessible to proteolysis, the regions that were inaccessible to tryptic digestion were found to correspond to hydrophobic domains on the protein surface. These results also suggest that the three-dimensional structures of these proteins remain largely intact upon adsorption to the hydrophobic surface.