Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis.

We report the discovery and characterization of a glycosylated bacterial ABC-type phosphate transporter isolated from the peripheral blood mononuclear cell (PBMC) fraction of patients with visceral leishmaniasis (VL). Three disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) of 19, 56 and 65 kDa, respectively, had been identified and their purity, apparent mass and pI established by SDS-PAGE and isoelectric focusing. Western blot analyses showed that the 9-O-acetylated sialic acid is linked via alpha2-->6 linkage to a subterminal N-acetylgalactosamine. For the 56 kDa protein, N- as well as O-glycosylations were demonstrated by specific glycosidase treatment and found to account for more than 9 kDa of the protein mass. The presence of sialic acids was further confirmed through thin layer chromatography, fluorimetric HPLC and electrospray ionization-mass spectrometry. The protein was identified by mass spectrometry and de novo sequencing of five tryptic fragments as a periplasmic ABC-type phosphate transporter of Pseudomonas aeruginosa. The amino acid sequences of the assigned peptides had 83-100% identity with the NCBI entry for a Pseudomonas transporter protein. Based on the recently reported X-ray structure of a human phosphate-binding protein, we predicted a 3D structural model for the 56 kDa protein using homology and threading methods. The most probable N- and O-glycosylation sites were identified by combinations of sequence motif-searching bioinformatics tools, solvent accessibility calculations, structural environment analyses and mass spectrometric data. This is the first reported glycosylation as well as sialylation of the periplasmic component of an ABC-type phosphate transporter protein and of one of few identified bacterial glycoproteins.

Biochemical determination of natural tumor-associated T-cell epitopes.

The knowledge of tumor-associated T-cell epitopes is important for the understanding of tumor biology and the development of cancer vaccines. We describe here a biochemical approach for the identification of tumor-associated T-cell epitopes. Peptides are extracted from immunoaffinity isolated MHC class I molecules of tumor cells and separated by HPLC. The HPLC fractions are then tested for biological activity of the peptides which are then sequenced by mass spectrometry. The tumor association of the identified T-cell epitopes is confirmed using synthetic analogs and T-cells of cancer patients.

Testing the role of gp96 as peptide chaperone in antigen processing.

gp96 is a 96-kDa glycoprotein of the endoplasmic reticulum that is believed to be involved in antigen processing as an intermediate carrier of peptides for presentation by major histocompatibility complex (MHC) class I molecules. This function implies that gp96 carries a large array of different peptides that represent the antigenicity of the cell and can serve all MHC class I molecules. So far, the evidence regarding these peptides is largely indirect and based on experiments where mice immunized with gp96 from tumor or virus-infected cells developed T cellular immune responses with the corresponding specificities. We analyzed by mass spectrometry peptides isolated from gp96 and found a number of different peptides derived from the proteins of different cellular compartments but mostly cytoplasm and nucleus. The sequences of these peptides provide information on the specificity of antigen processing and reveal structural requirements for binding to gp96 that only partially correspond to those of peptides presented by MHC class I molecules. The yield of peptides extracted from gp96 was far substoichiometric with an estimated occupancy of this chaperone of between 0.1% and 0.4%. These results strongly argue against a regular role for gp96 as a peptide chaperone in antigen processing.

Sequit: software for de novo peptide sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.

Peptide sequencing by mass spectrometry is gaining increasing importance for peptide chemistry and proteomics. However, available tools for interpreting matrix-assisted laser desorption/ionization post-source decay (MALDI-PSD) mass spectra depend on databases, and identify peptides by matching experimental data with spectra calculated from database sequences. This severely obstructs the identification of proteins and peptides not listed in databases or of variations, e.g. mutated proteins. The development of a new computer program for database-independent peptide sequencing by MALDI-PSD mass spectrometry is reported here. This computer program was validated by the determination of the correct sequences for various peptides including sequences listed in the sequence databases, but also for peptides that deviate from database sequences or are completely artificial. This strategy should substantially facilitate the identification of novel or variant peptides and proteins, and increase the power of MALDI-PSD analyses in proteomics.