Yanet Tambara

Effect of the position of a basic amino acid on C-terminal rearrangement of protonated peptides upon collision-induced dissociation

Internal rearrangement involving the loss of the C-terminal amino acid residue upon collision-induced dissociation (CID) or metastable decomposition was studied for protonated peptides. To investigate the structural characteristics of peptides responsible for this rearrangement, a series of synthetic peptides were prepared and subjected to B/E-linked scan or tandem mass spectrometric analyses using a four-sector instrument. The results showed that the position of a basic amino acid in the peptide sequence and its basicity have a significant influence on the rearrangement. Arginine (Arg) located at the n-1 position facilitates the rearrangement with about twice as many rearrangement ions as is observed for the other Arg-containing peptides. This can be attributed to the interaction of a positively charged guanidino group of Arg with its own carbonyl group via a salt bridge which is tightly formed in vacuo between a guanidino and carboxylate groups, the mechanism of which is analogous to that previously proposed for the formation of similar rearrangement ions observed in the spectra of metal-cationized peptides. This association would result in the facile attack of the C-terminal hydroxyl group on the penultimate carbonyl group, leading to the rearrangement. In addition, the rearrangement ion was observed both in metastable decomposition and high-energy CID spectra obtained by B/E-linked scan analyses without or with gas, respectively, but in a sequence dependent manner.

Sensitive Analysis of Oligosaccharides Derivatized with 4-Aminobenzoic Acid 2-(Diethylamino)ethyl Ester by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE) can be analyzed by ESI (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028−4031) and MALDI (Takao, T.; et al. Rapid Commun. Mass Spectrom. 1996, 10, 637−640) mass spectrometry. In this study, oligosaccharides derived from the enzymatic cleavage of the sugar chains of glycoproteins ribonuclease B, erythropoietin, and transferrin were subjected to ABDEAE derivatization, prior to analysis on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) for high-resolution mass measurement and a postsource decay (PSD) experiment. In the mass measurement of ABDEAE derivatives, quasi-molecular ion species have been observed in monoisotopic resolution using 2,5-dihydroxybenzoic acid as the matrix from spots that contain 50−200 fmol of sample; in the PSD analyses from the spots contained 500 fmol−1 pmol of sample, the predominant backbone ion series which covers the entire mass range for all the...

Expression in Escherichia coli of the lpdA gene, protein sequence analysis and immunological characterization of the P64k protein from Neisseria meningitidis

By making use of recombinant DNA technology it is possible to characterize meningococcal outer membrane proteins (OMPs) capable of stimulating a host immune response. The lpdA gene, which codes for an OMP (P64k) from Neisseria meningitidis, was cloned in Escherichia coli. The recombinant protein was recognized by sera from patients convalescing from meningococcal disease. The monoclonal antibodies obtained against the recombinant protein recognized the natural protein on a Western blot, and monoclonal antibody 114 was assayed in ELISA with a panel of 85 N. meningitidis strains. The protein was recognized in 81 strains (95.3%); the strains that were not recognized were neither epidemic nor isolated from systemic disease. The complete amino acid sequence of P64k was obtained by automatic sequencing and MS.

Structural characterization and biological implications of sulfated N-glycans in a serine protease from the neotropical moth Hylesia metabus (Cramer [1775]) (Lepidoptera: Saturniidae)

Contact with the urticating setae from the abdomen of adult females of the neo-tropical moth Hylesia metabus gives rise to an urticating dermatitis, characterized by intense pruritus, generalized malaise and occasionally ocular lesions (lepidopterism). The setae contain a pro-inflammatory glycosylated protease homologous to other S1A serine proteases of insects. Deglycosylation with PNGase F in the presence of a buffer prepared with 40% H2 (18)O allowed the assignment of an N-glycosylation site. Five main paucimannosidic N-glycans were identified, three of which were exclusively α(1-6)-fucosylated at the proximal GlcNAc. A considerable portion of these N-glycans are anionic species sulfated on either the 4- or the 6-position of the α(1-6)-mannose residue of the core. The application of chemically and enzymatically modified variants of the toxin in an animal model in guinea pigs showed that the pro-inflammatory and immunological reactions, e.g. disseminated fibrin deposition and activation of neutrophils, are due to the presence of sulfate-linked groups and not on disulfide bonds, as demonstrated by the reduction and S-alkylation of the toxin. On the other hand, the hemorrhagic vascular lesions observed are attributed to the proteolytic activity of the toxin. Thus, N-glycan sulfation may constitute a defense mechanism against predators.

Assessment of the Bordetella pertussis BpCNIC0311 Strain as a Producing Strain of Genetically Detoxified Toxoid (PTg), Filamentous Hemagglutinin (FHA) and Type 2 Pertactin (Prn2)

Pertussis is a vaccine preventable disease caused by the Gram-negative bacterium Bordetella pertussis ( B. pertussis ). Acellular vaccines as regard to whole cell vaccines are less reactogenic and very similar in efficacy. An increased incidence of B. pertussis infections in countries where acellular vaccines are widely used has questioned their effectiveness. In fact, the circulating strains differ from vaccine strains due to variations in protein sequences associated to the evolutionary process of B. pertussis . Furthermore, vaccine antigens change throughout the manufacturing process caused by chemical treatments for toxin inactivation. Both aspects should be considered when looking into the loss of efficacy of acellular vaccines. In this paper we study the performance of a new producing strain, BPCNIC0311. The strain expresses the genetically inactivated pertussis toxoid (PTg) and type 2 pertactin (Prn2), the most frequently occurring pertactin variant in clinical isolates. As a result, the new strain is able to grow stably at high cell density in a chemically defined culture medium. Similarly, the strain stably expresses high levels of PTg, FHA and Prn2 antigens which allow for suitable yields after purification. High purity and adequate physicochemical features of the antigens were obtained. The three components formulated in alum, as a stand-alone pertussis preparation, were highly immunogenic and induced a protective immune response in mice against the reference strain BP18323. It is concluded that the new strain can be used to obtain sufficient amounts of PTg, FHA and Prn2 for preclinical studies of an improved-updated acellular vaccine against whooping cough.