Evidence for long-range glycosyl transfer reactions in the gas phase

AbstractA long-range glycosyl transfer reaction was observed in the collision-induced dissociation Fourier transform (CID FT) mass spectra of benzylamine-labeled and 9-aminofluorene-labeled lacto-N-fucopentaose I (LNFP I) and lacto-N-difucohexaose I (LNDFH I). The transfer reaction was observed for the protonated molecules but not for the sodiated molecules. The long-range glycosyl transfer reaction involved preferentially one of the two L-fucose units in labeled LNDFH I. CID experiments with labeled LNFP I and labeled LNFP II determined the fucose with the greatest propensity for migration. Further experiments were performed to determine the final destination of the migrating fucose. Molecular modeling supported the experiments and reaction mechanisms are proposed

A human DNA editing enzyme homologous to the Escherichia coli DnaQ/MutD protein

Mammalian DNA polymerases alpha and beta lack 3' exonuclease activity and are unable to edit errors after DNA synthesis. However, editing exonucleases can be functions of separate polypeptides. We isolated a widely distributed DNA-specific 3' exonuclease from rabbit liver nuclei, sequenced tryptic peptides by mass spectrometry, and identified the corresponding human open reading frame. The protein expressed from the cloned human sequence exhibits 3' exonuclease activity. The human clone shares sequence homology with the editing function of the Escherichia coli DNA polymerase III holoenzyme, i.e., the DnaQ/MutD protein, and weakly with the editing 3' exonuclease domain of eukaryotic DNA polymerase epsilon. The gene maps to human chromosome 3p21.2-21.3. In a reconstituted human DNA repair system containing DNA polymerase beta and DNA ligase III-XRCC1, accurate rejoining of a 3' mismatched base residue at a single-strand break is dependent on addition of the exonuclease

The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis--preliminary findings

Proteomics-based studies offer a powerful complementary approach to DNA/RNA-based investigations and are now being applied to investigate aspects of many diseases including cancer. However, the heterogeneous nature of tissue samples often makes interpretation difficult. We have undertaken a study into the potential use of a novel laser capture microdissection (LCM) system to isolate cells of interest for subsequent proteomic analysis. Retrieval of selected cells is achieved by activation of a transfer film placed in contact with a tissue section, by a laser beam (30 or 60 microm diameter) which is focused on a selected area of tissue using an inverted microscope. The precise area of film targeted by the laser bonds to the tissue beneath it and these cells are then lifted free of surrounding tissue. Although the technique has been shown to be readily compatible with subsequent analysis of nucleic acids, little information is yet available regarding the application of protein-based analyses to the captured tissue. We report here preliminary data regarding the potential use of the LCM system in combination with two-dimensional electrophoresis to examine protein profiles of selected tissue areas. Electrophoretic profiles of proteins from normal and malignant renal tissue samples showed little change following LCM, nine selected proteins showed identical mass spectrometric sequencing profiles, and two selected proteins retained antigenicity. Dissection of epithelial tissue from a sample of normal human cervix resulted in enrichment of some proteins compared with analysis of the whole tissue. LCM will be a valuable adjunct to proteomic studies although further detailed validation is necessary