The Leishmania donovani secretome

Analysis of Leishmania-conditioned medium resulted in the identification of 151 proteins apparently secreted by the parasitic protozoan Leishmania donovani and suggested a vesicle-based secretion system.

The proteome of any cell or even any subcellular fraction remains too complex for complete analysis by one dimension of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hence, to achieve greater depth of coverage for a proteome of interest, most groups routinely subfractionate the sample prior to LC-MS/MS so that the material entering LC-MS/MS is less complex than the original sample. Protein and/or peptide fractionation methods that biochemists have used for decades, such as strong cation exchange chromatography (SCX), isoelectric focusing (IEF) and SDS-PAGE, are the most common prefractionation methods used currently. There has, as yet, been no comprehensive, controlled evaluation of the relative merits of the various methods, although some binary comparisons have been made. Here, we compare the most popular methods for fractionating samples at both the protein and peptide level, replicating all analyses to provide estimates of the variability in the analyses and controlling precisely for instrument time dedicated to each analysis, as well as directly measuring the recovery of protein or peptide from each fractionation procedure. For maximal proteome coverage, SDS-PAGE is very clearly the most effective method tested, with more than 90% of the entire data set found. When considering the amount of material recovered after each fractionation procedure, solution-based IEF and SCX performed similarly, with approximately 80% of the input being recovered.

No abstract

Understanding the role for DNA methylation in tumorigenesis has evolved from de®ning the location and extent of methylation in a variety of cancer-related genes to clarifying the functional and site-speci®c eects of aberrant methylation on gene expression. Our objectives were to characterize the functional eects of DNA methylation in the BRCA1 promoter and to clarify the functional status of the BRCA1 CRE (cAMP response element) motif. Luciferase reporter assays con®rm that an intact CRE is important for BRCA1 expression in transient transfections. Luciferase activities were decreased in constructs where the CRE recognition sequence was altered and when constructs were methylated in vitro. Gel mobility shift and competition assays identi®ed a DNA-protein complex recognizing the CRE motif that we were able to supershift using CREBspeci®c antibody. Furthermore this CRE is methylation sensitive, and we localized this methylation eect to a CpG dinucleotide within the BRCA1 CRE motif. The consequences of aberrant DNA methylation at speci®c transcription factor motifs, along with the multiple mutational events that can occur in a variety of essential genes such as BRCA1, paint a complex picture where both genetic and epigenetic changes contribute to tumour formation. Oncogene (2001) 20, 5331 ± 5340.