Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays.

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 µl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-ß-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique.

Ongoing genome reduction in Mycobacterium ulcerans.

Elucidation of the transmission, epidemiology, and evolution of Mycobacterium ulcerans, the causative agent of Buruli ulcer, is hampered by the striking lack of genetic diversity of this emerging pathogen. However, by using a prototype plasmid-based microarray that covered 10% of the genome, we found multiple genomic DNA deletions among 30 M. ulcerans clinical isolates of diverse geographic origins. Many of the changes appear to have been mediated by insertion sequence (IS) elements IS2404 and IS2606, which have high copy numbers. Classification of the deleted genes according to their biological functions supports the hypothesis that M. ulcerans has recently evolved from the generalist environmental M. marinum to become a niche-adapted specialist. The substantial genomic diversity, along with a prototype microarray that covered a small portion of the genome, suggests that a genome-wide microarray will make available a genetic fingerprinting method with the high resolution required for microepidemiologic studies.