Clinically relevant bioactivities of human galectins (adhesion/growth-regulatory galactoside-specific lectins) inspired the design of peptides as new tools to elicit favorable effects (e.g., in growth control) or block harmful binding (e.g., in tissue invasion). To obtain the bioinspired lead compounds, we combined a proteolytic fragmentation approach without/with ligand contact (excision) with mass spectrometric identification of affinity-bound protein fragments, using galectin-1 and -3 as models. Two peptides from the carbohydrate recognition domains were obtained in each case in experimental series rigorously controlled for specificity, and the [157-162] peptide of galectin-3 proved to be active in blocking lectin binding to a neoglycoprotein and to tumor cell surfaces. This approach affords peptide sequences for structural optimization and intrafamily/phylogenetic galectin comparison at the binding-site level with a minimal requirement of protein quantity, and it is even amenable to mixtures.

Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine-specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3-NT-specific antibody, and have synthesized a series of tyrosine-nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr-430 had been previously identified upon reaction with peroxynitrite17. The determination of antibody-binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr-430, Tyr-421, Tyr-83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N-terminal to the nitration site. The highest affinity to the anti-3NT-antibody was found for the PCS peptide comprising the Tyr-430 nitration site with a K(D) of 60 nM determined for the peptide, PCS(424-436-Tyr-430NO(2)); in contrast, PCS peptides nitrated at Tyr-421 and Tyr-83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody-bound peptides and affinity-MS analysis revealed highest affinity to the antibody for tyrosine-nitrated peptides that contained positively charged amino acids in the N-terminal sequence to the nitration site. Remarkably, similar N-terminal sequences of tyrosine-nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil-cationic protein.

A new methodology using hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of protein-peptide interactions is reported. The method, based on our previously established approach, protein ligand interaction by mass spectrometry, titration, and H/D exchange (PLIMSTEX)[J. Am. Chem. Soc.2003, 125, 5252-5253], makes use of a dilution strategy (dPLIMSTEX) for HDX, using the mass of the peptide ligand as readout. We employed dPLIMSTEX to study the interaction of calcium-saturated calmodulin with the opioid peptide beta-endorphin as a model system; the affinity results are in good agreement with those from traditional PLIMSTEX and with literature values obtained by using other methods. We show that the dPLIMSTEX method is feasible to quantify an antigen-antibody interaction involving a 3-nitrotyrosine modified peptide in complex with a monoclonal anti-nitrotyrosine antibody. A dissociation constant in the low nanomolar range was determined, and a binding stoichiometry of antibody/peptide of 1:2 was confirmed. In addition, we determined that the epitope in the binding interface contains a minimum of five amino acids. The dPLIMSTEX approach is a sensitive and powerful tool for the quantitative determination of peptide affinities with antibodies, complementary to conventional immuno-analytical techniques.

Formation and accumulation of fibrillar plaques and aggregates of beta-amyloid peptide (Abeta) in brain have been recognized as characteristics of Alzheimer's disease (AD). Oligomeric aggregates of Ass are considered critical intermediates leading to progressive neurodegeneration; however, molecular details of the oligomerization and aggregation pathway and the structures of Abeta-oligomers are hitherto unclear. Using an in vitro fibril formation procedure of Abeta(1-40), beta-amyloid aggregates were prepared and insoluble aggregates separated from soluble products by centrifugation. In this study, ion mobility mass spectrometry (IM-MS) was applied in combination with electron paramagnetic resonance spectroscopy (EPR) to the identification of the components of Abeta-oligomers, and to their structural and topographical characterization. The formation of Abeta-oligomers and aggregates was monitored by gel electrophoresis, and Abeta-oligomer bands were identified by in-gel tryptic digestion and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) to consist predominantly of Abeta(1-40) peptide. First, ion mobility-MS studies of soluble Abeta-aggregates prepared by incubation for 5 days were performed on a quadrupole time-of-flight mass spectrometer and revealed (1) the presence of at least two different conformational states, and (2), the formation of Met-35 oxidized products. For estimation of the size of Abeta-aggregates using EPR spectroscopy, a modified Abeta(1-40) peptide containing an additional N-terminal cysteine residue was prepared, and a 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy radical spin label derivative (IPSL) was coupled by S-alkylation. The EPR spectra of the spin-labeled Cys-Abeta(1-40) oligomers were matched with spectra simulations using a multi-component simulation strategy, resulting in complete agreement with the gel electrophoresis results.

The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision-mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose,(2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrP(C)) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision-MS, with complete suppression of nonspecific bPrP binding.

An antigenic mimic of the Ebola glycoprotein was synthesized and tested for its ability to be recognized by an anti-Ebola glycoprotein antibody. Epitope-mapping procedures yielded a suitable epitope that, when presented on the surface of a nanoparticle, forms a structure that is recognized by an antibody specific for the native protein. This mimic-antibody interaction has been quantitated through ELISA and QCM-based methods and yielded an affinity (K(d)= 12 x 10(-6) M) within two orders of magnitude of the reported affinity of the native Ebola glycoprotein for the same antibody. These results suggest that the rational design approach described herein is a suitable method for the further development of protein-based antigenic mimics with potential applications in vaccine development and sensor technology.

In order to keep subscribers up-to-date with the latest developments in their field, John Wiley & Sons are providing a current awareness service in each issue of the journal. The bibliography contains newly published material in the field of mass spectrometry. Each bibliography is divided into 11 sections: 1 Reviews; 2 Instrumental Techniques & Methods; 3 Gas Phase Ion Chemistry; 4 Biology/Biochemistry: Amino Acids, Peptides & Proteins; Carbohydrates; Lipids; Nucleic Acids; 5 Pharmacology/Toxicology; 6 Natural Products; 7 Analysis of Organic Compounds; 8 Analysis of Inorganics/Organometallics; 9 Surface Analysis; 10 Environmental Analysis; 11 Elemental Analysis. Within each section, articles are listed in alphabetical order with respect to author (4 Weeks journals - Search completed at 29th. June 2007).

Physiological β-amyloid autoantibodies (Aβ-autoantibodies) are currently investigated as potential diagnostic and therapeutic tools for Alzheimer's disease (AD). In previous studies, their determination in serum and cerebrospinal fluid (CSF) using indirect ELISA has provided controversial results, which may be due to the presence of preformed Aβ antigen-antibody immune complexes. Based on the epitope specificity of the Aβ-autoantibodies, recently elucidated in our laboratory, we developed (a) a sandwich ELISA for the determination of circulating Aβ-IgG immune complexes and (b) an indirect ELISA for the determination of free Aβ-autoantibodies. This methodology was applied to the analysis of serum samples from healthy individuals within the age range of 18 to 89 years. Neuropsychological examination of the participants in this study indicated non-pathological, age-related cognitive decline, revealed especially by tests of visual memory and executive function, as well as speed-related tasks. The ELISA serum determinations showed significantly higher levels of Aβ-IgG immune complexes compared to free Aβ-autoantibodies, while no correlation with age or cognitive performance of the participants was found.

Humanin (HN) is a linear 24-aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß-amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1-40) and characterization of the interaction structure through a molecular modeling study. Wild-type HN and HN-sequence mutations were synthesized by SPPS and the HPLC-purified peptides characterized by MALDI-MS. The interaction epitopes between HN and Aß(1-40) were identified by affinity-MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity-bound peptides. The affinity-MS analyses revealed HN(5-15) as the epitope sequence of HN, whereas Aß(17-28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1-40) and by ELISA with Aß(1-40) and Aß-partial sequences as ligands to immobilized HN. The specificity and affinity of the HN-Aß interaction were characterized by direct ESI-MS of the HN-Aß(1-40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K(D) of the complex of 610 nm. A molecular dynamics simulation of the HN-Aß(1-40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1-40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN.

The modification of ubiquitin to defined oligo-ubiquitinated conjugates has received considerable interest due to the finding that isomeric oligo-ubiquitin conjugates exhibit distinct differences in their biochemical functions, depending on the specific lysine-epsilon-amino linkage used for conjugate formation. Here, we report the design and development of a thioether linkage-based approach for the synthesis of oligo-ubiquitin conjugates with lysine-specific branching by thioether ligation of a linear ubiquitin peptide containing a C-terminal cysteine residue as the "donor" component, with a corresponding lysine-epsilon-amino-branched haloacyl-activated ubiquitin "acceptor" peptide. This approach was successfully used for the synthesis of a lysine-63-linked diubiquitin conjugate by ligation of the modified ubiquitin(1-52)-Cys- donor peptide to the N-terminal Arg-54 residue of the branched Lys-63-linked acceptor peptide, ubiquitin(54-76)(2). Advantages of the present approach are as follows:(i) the conjugation reaction is performed in solution using suitable preformed donor ubiquitin peptides with a C-terminal Cys residue, and (ii) different corresponding N-chloroacetylated ubiquitin acceptor peptides containing the branched Lys residue are employed, providing broad applicability to the preparation of isomeric oligo-ubiquitin conjugates. The Lys-63-diubiquitin conjugate 7 described here was purified by semipreparative HPLC, and its structure and homogeneity ascertained by HPLC and high-resolution MALDI and electrospray-mass spectrometry. CD spectra and molecular modeling indicate a conformationally stable structure of the conjugate with spatial separation of the ubiquitin parts of the Lys-63 linkage. Moreover, the activity of the thioether-linked diubiquitin conjugate was ascertained by in vitro autoubiquitination assay. These results indicate the feasibility of this approach for the preparation of functional oligo-ubiquitin conjugates.

Alzheimer's disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in brain that are directly involved in AD pathogenesis. The major component of AD plaques is beta-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, beta-and gamma-secretase acting at the N- and C- terminal cleavage site, respectively. In this study we have prepared polypeptides comprising the carboxy-terminal and transmembrane sequences of APP, by bacterial expression and chemical synthesis, as substrates for studying the C-terminal processing of APP and its interaction with the gamma-secretase complex. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used as a major tool for structure analysis. Immunisation of transgenic mouse models of AD with Abeta42 has been recently shown to be effective to inhibit and disaggregate Abeta-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects has been as yet unclear. Using proteolytic epitope excision from immune complexes in combination with FT-ICR-MS, we identified the epitope recognised by the therapeutically active antibody as the N-terminal Abeta(4-10) sequence; this soluble, nontoxic epitope opens new lead structures for AD vaccine development. A monoclonal antibody (Jonas; JmAb) directed against the cytosolic APP domain was used in studies of APP biochemistry and metabolism. Here we report the identification of the epitope recognised by the JmAb, using the combination of epitope excision and peptide mapping by FT-ICR-MS. The epitope was determined to be located at the C-terminal APP(740-747) sequence; it was confirmed by ELISA binding assays and authentic synthetic peptides and will be an efficient tool in the development of new specific vaccines. These results demonstrate high-resolution FT-ICR-MS as a powerful method for characterising biochemical pathways and molecular recognition structures of APP.

Amyloid plaques associated to Alzheimer's disease present a high content of zinc ions. We previously showed that the N-terminal region of the amyloid peptide Abeta constitutes an autonomous zinc-binding domain. This region encompasses the previously identified epitope Abeta(4-10) targeted by antibodies capable to reduce amyloid deposition, but the influence of Abeta/Zn binding on the epitope recognition remains unknown. We demonstrate here the effect of Zn2+ ions on the recognition of peptides sharing the sequence of the Abeta N-terminal domain, by two monoclonal antibodies recognizing the beta-amyloid(4-10) epitope. The presence of Zn2+, but not of other cations, increased the recognition of the (1-16) peptide, while it was without effect on the recognition of the (1-10) peptide. These findings show a zinc-induced conformational change of the (1-16)-N-terminal region of AP3, which results in a better accessibility of the Abeta(4-10) epitope to the anti-Abeta antibodies, and suggest a role of zinc in epitope-based vaccination approaches.

Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing two chemotherapeutic agents, daunorubicin and methotrexate, coupled to the GnRH-III decapeptide, which served as a targeting moiety. This represents a possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of anticancer drug-peptide bioconjugates. The multifunctional bioconjugates were prepared according to two drug design approaches recently developed by our group. Both bifunctional GnRH-III derivatives,[(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) and [(8)Lys(Lys)]-GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys(Lys)-Pro-Gly-NH(2)), contain two free amino groups suitable for the attachment of two anticancer drugs, such as methotrexate and daunorubicin. The drugs were chosen with respect to their different mechanisms of action, with the goal of increasing the antitumor effect of the bioconjugates. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their in vitro stability/degradation in human serum and in the presence of rat liver lysosomal homogenate was investigated by liquid chromatography in combination with mass spectrometry. The influence of the multifunctional bioconjugates on the cell adhesion and cell proliferation was studied on Mono Mac 6 human leukemic monocytes. It was found that (1) all synthesized bioconjugates had in vitro cytostatic effect;(2) they were stable in human serum for at least 24 h;(3) they were hydrolyzed in the presence of lysosomal homogenate and (4) they exerted a moderate cell-cell adhesion inducing effect. These results demonstrate that multifunctional bioconjugates containing two different anticancer drugs attached to the same GnRH-III targeting moiety could be successfully prepared and resulted in higher in vitro cytostatic effect than the monofunctional bioconjugates containing either methotrexate or daunorubicin, in particular on HT-29 human colon cancer cells.

Modification of ubiquitin, a key cellular regulatory polypeptide of 76 amino acids, to polyubiquitin conjugates by lysine-specific isopeptide linkage at one of its seven lysine residues has been recognized as a central pathway determining its biochemical properties and cellular functions. Structural details and differences of distinct lysine-isopeptidyl ubiquitin conjugates that reflect their different functions and reactivities, however, are only partially understood. Ion mobility spectrometry (IMS) combined with mass spectrometry (MS) has recently emerged as a powerful tool for probing conformations and topology involved in protein interactions by an electric field-driven separation of polypeptide ions through a drift gas. Here we report the conformational characterization and differentiation of Lys63- and Lys48-linked ubiquitin conjugates by IMS-MS. Lys63- and Lys48-linked di-ubiquitin conjugates were prepared by recombinant bacterial expression and by chemical synthesis using a specific chemical ligation strategy, and characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry, circular dichroism spectroscopy, and molecular modeling. IMS-MS was found to be an effective tool for the identification of structural differences of ubiquitin complexes in the gas phase. The comparison of collision cross-sections of Lys63- and Lys48-linked di-ubiquitin conjugates showed a more elongated conformation of Lys63-linked di-ubiquitin. In contrast, the Lys48-linked di-ubiquitin conjugate showed a more compact conformation. The IMS-MS results are consistent with published structural data and a comparative molecular modeling study of the Lys63- and Lys48-linked conjugates. The results presented here suggest IMS techniques can provide information that complements MS measurements in differentiating higher-order polyubiquitins and other isomeric protein linkages.

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin),(4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum,(2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect,(2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.

Human cystatin C (hCC), like many other amyloidogenic proteins, has been shown to form dimers by exchange of subdomains of the monomeric protein. Considering the model of hCC fibrillogenesis by propagated domain swapping, it seems possible that inhibition of this process should also suppress the entire process of dimerization and fibrillogenesis which leads to specific amyloidosis (hereditary cystatin C amyloid angiopathy (HCCAA)). It was reported that exogenous agents like monoclonal antibody against cystatin C are able to suppress formation of cystatin C dimers. In the effort to find a way of controlling the cystatin fibrillization process, the interactions between monoclonal antibody Cyst-13 and cystatin C were studied in detail. The present work describes the determination of the epitope of hCC to a monoclonal antibody raised against cystatin C, Cyst-13, by MALDI mass spectrometry, using proteolytic excision of the immune complex. The shortest epitope sequence was determined as hCC(107-114). Affinity studies of synthetic peptides revealed that the octapeptide with epitope sequence does not have binding ability to Cyst-13, whereas its longer counterpart, hCC(105-114), binds the studied antibody. The secondary structure of the peptides with epitope sequence was studied using circular dichroism and NMR spectroscopy.

Bioconjugates containing the GnRH-III hormone decapeptide as a targeting moiety are able to deliver chemotherapeutic agents specifically to cancer cells expressing GnRH receptors, thereby increasing their local efficacy while limiting the peripheral toxicity. However, the number of GnRH receptors on cancer cells is limited and they desensitize under continuous hormone treatment. A possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of the bioconjugates would be the attachment of more than one chemotherapeutic agent to one GnRH-III molecule. Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing GnRH-III as a targeting moiety and daunorubicin as a chemotherapeutic agent. Two different drug design approaches were pursued. The first one was based on the bifunctional [(4) Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) containing two lysine residues in positions 4 and 8, whose ε-amino groups were used for the coupling of daunorubicin. In the second drug design, the native GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was used as a scaffold; an additional lysine residue was coupled to the ε-amino group of (8) Lys in order to generate two free amino groups available for conjugation of daunorubicin. The in vitro stability/degradation of all synthesized compounds was investigated in human serum, as well as in the presence of rat liver lysosomal homogenate. Their cellular uptake was determined on human breast cancer cells and the cytostatic effect was evaluated on human breast, colon and prostate cancer cell lines. Compared to a monofunctional compound, both drug design approaches resulted in multifunctional bioconjugates with increased cytostatic effect. © 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2011.

The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.

PURPOSE To investigate the expression of alpha-Gal or unidentified non-Gal antigens in pig corneal endothelial cells and keratocytes, we performed the qualitative and quantitative analysis by using mass spectrometry. METHODS The N-glycans from common adult pig corneal endothelial cells and keratocytes cultured in vitro were directly analyzed by using mass spectrometric approaches. In addition, immunochemical staining was added to confirm the non-Gal antigen expression in pig corneal cells. RESULTS Totally, 34 of the sialylated N-glycans from pig corneal endothelial cells and 27 from pig keratocytes were identified and observed to contain nonhuman sialic acid, NeuGc as well as NeuAc. In addition, we were able to detect 25 of alpha-galactosylated N-glycan structures (22.2% of total) from the pig corneal endothelial cells and 18 of that (17.5% of total) from the pig keratocytes by using mass spectrometric approaches. On immunofluorescent staining, the expression of sialylated glycans was also observed. CONCLUSIONS As well as alpha-Gal epitopes, several promising non-Gal antigens were widely expressed on both pig corneal endothelial cells and keratocytes. The detailed structural information of the alpha-Gal and non-Gal epitopes would be a tremendous value to develop a new strategy for the successful corneal xenotransplantation in future.

In the last years, intracellular protein degradation by the proteasome has become a focus area of scientific interest. Here, we describe a proteomics approach for the molecular mapping of the constituents of the proteolytically active core particle, the constitutive 20S proteasome from mouse intestine. In addition to the proteomics workflow widely used for protein isolation, gel electrophoretic separation, in-gel digestion, and UV-MALDI mass spectrometry, high-resolution Fourier transform ion cyclotron resonance mass spectrometry using infrared-MALDI ionisation (IR-MALDI FTICR-MS) has been employed as an efficient method for protein identification by peptide mass fingerprint. The 20S proteasome subunits alpha1-alpha7 and beta1-beta7 were completely and unambiguously identified. In addition to subunits beta1 and beta2, the corresponding inducible subunits being part of the immuno-proteasome were identified. The subunit beta5i was found to completely replace the corresponding constitutive subunit, suggesting a high proteolytic activity of the intestinal proteasome leading to increased production of antigenic peptides. The high mass accuracy in the low ppm range and resolution of FTICR-MS provide direct identifications of individual proteins as mixtures such as components resulting from incomplete electrophoretic separation. In addition, the comparison of UV- and IR-MALDI FTICR-MS may provide details of fragmentation and rearrangement reactions that may occur under UV-MALDI ionisation conditions.

The aim of this chapter is to provide a strategy for mapping linear antibody epitopes of protein antigens in order to discover candidates for vaccines or diagnostic tests. A set of overlapping peptides was designed and synthesised based upon a known amino acid sequence of the target protein, virulence-associated protein A (VapA) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals.The peptides were biotinylated and used in an ELISA to screen immune sera from foals. These biotinylated peptides were coated directly onto micro titre plates that had been pre-coated with NeutrAvidin. A linear B-cell epitope was identified by a universal recognition of sera to the synthetic peptides which corresponds to a particular fragment of the VapA protein.

The ability to accurately characterize an epitope on an antigen is essential to understand the pathogenesis of an infectious material, and for the design and development of drugs and vaccines. Emergence of a new contagious microbial or viral variant necessitates the need for robust identification and characterization of the antigenic determinant. Recent advances have made mass spectrometry (MS) a robust and sensitive analytical tool with high mass accuracy. The use of MS to characterize peptides and proteins has gained popularity in the research arena involving protein-protein interactions. Combining the modern mass spectrometric principles of protein-protein interaction studies with the classical use of limited proteolysis, a linear epitope on a peptide or a protein antigen can be accurately mapped in a short time, compared with other traditional techniques available for epitope mapping. Additionally, complete MS analyses can be achieved with very little sample consumption. Here we discuss the overall approach to characterize the detailed interaction between a linear antigen (either a peptide or a protein antigen) and its corresponding monoclonal antibody by using MS. The steps involved in epitope excision, epitope extraction, and indirect immunosorption are outlined thoroughly. Conditions required for MS analysis using either matrix assisted laser desorption ionization (MALDI) or electrospray ionization (ESI) sources are summarized, with special emphasis on the experimental protocols.

The use of antigen fragments generated by specific proteolytic cleavage is a relatively simple "library" approach for epitope mapping in which possible overlapping fragments are screened with the antibody on Western blots. Proteolytic fragmentation with numerous proteases having different cleavage specificites can be carried out on native and denaturated proteins, generating a small and large number of fragments, respectively. To determine the antigenic site of a monoclonal antibody, we have examined the limited proteolytic digestion of the transducin alpha -subunit with four different proteases and detected antibody binding to fragments by Western blot. Using this approach, the epitope for this antibody was localized within the amino-terminal 17 residues of transducin alpha -subunit.

Progress is reviewed towards the development of a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness and throughput of proteomic measurements based upon the use of high performance separations and mass spectrometry. The approach uses high accuracy mass measurements from Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate peptide 'accurate mass tags'(AMTs) produced by global protein enzymatic digestions for a specific organism, tissue or cell type from 'potential mass tags' tentatively identified using conventional tandem mass spectrometry (MS/MS). This provides the basis for subsequent measurements without the need for MS/ MS. High resolution capillary liquid chromatography separations combined with high sensitivity, and high resolution accurate FTICR measurements are shown to be capable of characterizing peptide mixtures of more than 10(5) components. The strategy has been initially demonstrated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, high sensitivity, and the capability for stableisotope labeling methods for precise relative protein abundance measurements.Abbreviations: LC, liquid chromatography; FTICR, Fourier transform ion cyclotron resonance; AMT, accurate mass tag; PMT, potential mass tag; MMA, mass measurement accuracy; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ppm, parts per million.

Human beta-amyloid (Abeta) is believed to be one of the main components of Alzheimer's disease, so reduction of Abeta is considered a key therapeutic target. Using Agrobacterium-mediated nuclear transformation, we generated transgenic tomatoes for Abeta with tandem repeats. Integration of the human Abeta gene into the tomato genome and its transcription were detected by PCR and Northern blot, respectively. Expression of the Abeta protein was confirmed by western blot and ELISA, and then the transgenic tomato line expressing the highest protein level was selected for vaccination. Mice immunized orally with total soluble extracts from the transgenic tomato plants elicited an immune response after receiving a booster. The results indicate that tomato plants may provide a useful system for the production of human Abeta antigen.

Vanadyl (VO) porphyrins and sulfur-containing vanadyl (VOS) porphyrins of a wide carbon number range (C(26) to C(52)) and Z-number range (-28 to -54) were detected and identified in a petroleum asphaltene by atmospheric pressure photonionization (APPI) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). APPI provides soft ionization of asphaltene molecules (including VO and VOS porphyrins), generating primarily molecular ions (M(+.)). The ultra-high mass resolving power (m/Delta m(FWHM) approximately 500 K) of FTICR-MS enabled resolution and positive identification of elemental formulae for the entire family of VO and VOS porphyrins in a complicated asphaltene matrix. Deocophylerythro-etioporphyrin (DPEP) is found to be the most prevalent structure, followed by etioporphyrins (etio)- and rhodo (benzo)-DPEP. The characteristic Z-distribution of VO porphyrins suggests benzene and naphthene increment in the growth of porphyrin ring structures. Bimodal carbon number distributions of VO porphyrins suggest possible different origins of low and high molecular weight species. To our knowledge, the observation of VOS porphyrins in a petroleum product has not previously been reported. The work is also the first direct identification of the entire vanadyl porphyrin family by ultra-high resolution mass spectrometry without chromatographic separation or demetallation.

Several polypeptides comprising the carboxy-terminal domain of the 1-amyloid precursor protein (cAPP) were prepared by solid phase peptide synthesis, and employed as antigens for the determination of the epitopes recognised by anti-cAPP antibodies. Selective proteolytic epitope-excision and -extraction on the immobilised immune complexes, in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) were used as major methods for epitope identification. The epitope recognised by a polyclonal anti-cAPP antibody (36-BO) was identified as APP(727-737), a sequence close to the APP transmembrane region. In contrast, the epitope recognised by a monoclonal anti-cAPP antibody (Jonas-mAb) was identified at APP(740-747) to be located more remote from the transmembrane region. The two adjacent, yet distinct epitopes recognised by two different antibodies should provide efficient tools for (i), molecular diagnostic applications, and (ii), the study of intracellular processing pathways of APP relevant to Alzheimer's disease, utilising suitable mass spectrometric and molecular imaging approaches.