Q Bioanalytic (Germany)

Bacteria belonging to the genus Vibrio are very common to marine and estuarine environments and are found in association with marine plants and animals. Vibrio parahaemolyticus strains that produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), toxigenic strains of Vibrio cholerae belonging to the serogroups O1 and O139, and Vibrio vulnificus are regarded as important food-borne pathogens, which represent a serious and growing public health hazard. In this study, we established and validated real-time PCR assays for the detection of enteropathogenic Vibrio strains. In a first step, seafood is investigated for the presence of the three Vibrio species. In case of detection of V. cholerae or V. parahaemolyticus, samples are tested for the presence of the cholera toxin gene (ctxA) or tdh/trh genes, respectively, in a second step. All PCR analyses were performed with the same cycling program. Primer/probe sets were thoroughly tested for limit of detection, inclusivity, exclusivity and performance in the matrix. In an interlaboratory study, kits based on these primer/probe sets were successfully tested with cultural and DNA samples.

Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.

Abstract Originally published in: Analysing Gene Expression. Edited by Stefan Lorkowski, Paul Cullen. Copyright © 2003 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30488‐2The sections in this article areIntroductionHow does mass spectrometry work?Ionisation of moleculesDetection of ionsAppearance of spectraModes of operationProteomic mass spectrometryData analysis tools for mass spectroscopic experimentsProteomics servers and databasesAn example: Identification of an isolated and unknown proteinIs quantification possible with mass spectrometry?Analysis of protein modificationsPhosphorylationGlycosylationAccess to protein tertiary structureAnalysis of intact proteins and non‐covalent protein complexesAffinity mass spectrometryConclusion