O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

Application of proteomics methods for pathogen discovery.

Proteomics have been used widely to study proteins in complex materials such as cells, body fluids, tissues, and organisms. Application of advance proteomic techniques for the characterization of disease-specific proteins may provide information for the detection of potential infectious agents. In this report, two proteomics techniques, a two-dimensional differential gel electrophoresis (2D-DIGE) and a one-dimensional gel electrophoresis and one-dimensional liquid chromatography coupled with mass spectrometry (GeLC-MS/MS), were applied for investigating viral proteins from cultured cells inoculated with a clinical sample. The 2D-DIGE method identified five viral proteins of vaccinia virus that are only present in infected cells, these results are in agreement with findings determined by genome based methods. The GeLC-MS/MS method identified eight vaccinia virus proteins out of 428 proteins detected in the sample. These results demonstrate that proteomic techniques can be used effectively for the detection of infectious agents. Given that the methods are capable of applying to proteins without a prior knowledge of the pathogen present, proteomics has a potential of being developed as a molecular tool for pathogen discovery, and disease diagnosis of emerging infectious diseases and for bioterrorism defense.

N-terminal derivatization of peptides with isothiocyanate analogues promoting Edman-type cleavage and enhancing sensitivity in electrospray ionization tandem mass spectrometry analysis.

N-terminal derivatization of peptides with Edman's reagent, phenyl isothiocyanate (PITC), promotes gas-phase Edman cleavage that yields abundant complementary b(1) and y(n-1) ion pairs by tandem mass spectrometry (MS/MS). The formation of b(1) ions can be utilized as a mass tag to enhance the interpretation of MS/MS spectra and increase the confidence of peptide identification during mass spectrometry analysis. Derivatization of tryptic peptides with another isothiocyanate analogue, 4-sulfophenyl isothiocyanate, also produces signature ions resulting from Edman cleavage and facilitates peptide sequencing on linear or branched peptides. The limitation of these derivatizations, however, is reduced MS signal intensities of modified peptides, due presumably to the tags themselves. Here we have demonstrated that several other isothiocyanate analogues bearing basic moieties can derivatize peptides and significantly improve the MS sensitivity of tagged analytes, while promoting Edman fragmentation and maintaining other sequence fragments as well.