Alpha actinin is specifically recognized by Multiple Sclerosis autoantibodies isolated using an N-glucosylated peptide epitope.

Sophisticated approaches have recently led to the identification of novel autoantigens associated with Multiple Sclerosis (MuS), e.g. neurofascin, contactin, CNPase, and other T-cell receptor membrane anchored proteins. These putative antigens, although differing from the conventional myelin derivatives, are conceptually based on an animal model of experimental autoimmune encephalomyelitis. In this report we describe the identification of putative antigens based on their recognition by autoantibodies isolated from MuS patient serum. In a previous work from this laboratory we have shown that a peptide probe, named CSF114(Glc), specifically identifies serum autoantibodies in a subset of MuS patients, representing ∼30% of the patient population. The autoantibodies, purified from MuS patients' sera (six), through CSF114(Glc) affinity chromatography, detected three immunoreactive protein bands present in the rat brain. Proteomic analysis of the immunoreactive bands, involving MALDI and MS/MS techniques, revealed the presence of four proteins distinguishable by their mass: alpha fodrin, alpha actinin 1, creatine kinase, and CNPase. The immunoreactive profile of these rat brain proteins was compared with that of commercially available standard proteins by challenging against either CSF114(Glc) purified MuS autoantibodies, or monoclonal antibodies. Further discrimination among the rat brain proteins was provided by the following procedure: whereas monoclonal antibodies recognized all rat brain proteins, isolated MuS specific antibodies recognize only alpha actinin 1 as a putative antigen. In fact, alpha actinin 1 displayed a robust immunoreactive response against all MuS patients' sera examined, whereas the other three bands were not consistently detectable. Thus, alpha actinin 1, a cytoskeleton protein implicated in inflammatory/degenerative autoimmune diseases (lupus nephritis and autoimmune hepatitis) might be regarded as a novel MuS autoantigen, perhaps a prototypic biomarker for the inflammatory/degenerative process typical of the disease.

Conventional and microwave-assisted SPPS approach: a comparative synthesis of PTHrP(1-34)NH(2).

Attracted by the possibility to optimize time and yield of the synthesis of difficult peptide sequences by MW irradiation, we compared Fmoc/tBu MW-assisted SPPS of 1-34 N-terminal fragment of parathyroid hormone-related peptide (PTHrP) with its conventional SPPS carried out at RT. MWs were applied in both coupling and deprotection steps of SPPS protocol. During the stepwise elongation of the resin-bound peptide, monitoring was conducted by performing MW-assisted mini-cleavages and analyzing them by UPLC-ESI-MS. Identification of some deletion sequences was helpful to recognize critical couplings and as such helped to guide the introduction of MW irradiations to these stages.

Detection of honeybee venom in envenomed tissues by direct MALDI MSI.

A new analytical approach using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) for the study of honeybee venom is shown. In vitro and in vivo models simulating the bee sting have been developed using live honeybees and, as the envenomation sites, pig ears and rat legs; MALDI MSI has been used to map, over time, the diffusion and distribution of three venom allergens (Api m 1, Api m 4, and Api m 6) and two venom toxins (apamine and mast cell degranulating peptide). In conjunction with other classical biochemical techniques and high resolution mass spectrometry (HRMS), structural data have been obtained that contribute to current understanding of honeybee venom composition. Initial data have also been obtained demonstrating the feasibility of mapping the organism's response to the sting. The opportunity to monitor venom diffusion and the organism's response at the same time might open new pathways for in vivo preclinical studies in designing and testing new venom immunotherapy (VIT).

Not only cuticular lipids: first evidence of differences between foundresses and their daughters in polar substances in the paper wasp Polistes dominulus.

Several studies have shown that differences in the relative abundance of cuticular hydrocarbons occur between reproductives and non-reproductives in many social insects. These differences also exist between Polistes dominulus foundresses and their first emerged daughters (usually indicated as workers), but they gradually disappear when workers from orphaned colonies develop their ovaries and assume a reproductive role in the colony. However, hydrocarbons are not the exclusive components of cuticular layer of wasps. Mass-spectrometry analysis of cuticular methanol extracts from Polistes paper wasps showed a complex pattern of polar substances, partly or totally proteinaceous in nature. We found that these compounds, ranging from 918 to 2679 Da, showed a clear caste differentiation between foundresses and their first emerged daughters (usually indicated as "workers"), both in queen-right and in orphaned colonies. Conversely to hydrocarbons, workers from orphaned colonies maintain a significant difference from foundresses in the pattern of the medium molecular weight (MW) polar compounds obtained by MALDI-TOF. On the basis of such results we hypothesize that a reliable cue to identify foundresses from daughters, and not only their reproductive status, may exist in Polistes wasps. Although the great majority of previous work on social insect communication has focused on cuticular hydrocarbons, our findings suggest that the medium MW component of cuticular substances may be involved in recognition.

Comparison of the medium molecular weight venom fractions from five species of common social wasps by MALDI-TOF spectra profiling.

The average spectral profiles and the exact mass weight (MW) of biomolecules present in the medium fraction (from 900 to 3000 Da) of the venom of five social wasps (three European and one North American Polistes and the European hornet Vespa crabro) were determined by matrix assisted laser desorption ionization time of flight (MALDI-TOF) MS. Data were obtained analyzing the venom of single specimens (N = 46) and elaborated with the ClinProTools 2.0 (CPT) software to search for differences among the five species examined. Interesting differences in the spectral profiles were found, allowing the discrimination of venoms belonging to the different species, and their possible use as a quality control method in venom immunotherapy (VIT) for allergic patients.

Dominulin A and B: two new antibacterial peptides identified on the cuticle and in the venom of the social paper wasp Polistes dominulus using MALDI-TOF, MALDI-TOF/TOF, and ESI-ion trap.

Two new antibacterial peptides, denominated as Dominulin A and B, have been found on the cuticle and in the venom of females of the social paper wasp Polistes dominulus. The amino acidic sequence of the two peptides, determined by mass spectrometry, is INWKKIAE VGGKIL SSL for Dominulin A (MW = 1854 Da) and INWKKIAEIGKQVL SAL (MW = 1909 Da) for Dominulin B. Their presence on the cuticle was confirmed using MALDI-TOF by means of micro-extractions and direct analyses on body parts. The presence in the venom and the primary structure of the dominulins suggest their classification in the mastoparans, a class of peptides found in the venom of other Aculeate hymenoptera. Their antimicrobial action against Gram+ and Gram- bacteria fits in the range of the best natural antimicrobial peptides. Dominulins can represent an important defense of the colony of Polistes dominulus against microbial pathogens.