Protein staining influences the quality of mass spectra obtained by peptide mass fingerprinting after separation on 2-d gels. A comparison of staining with coomassie brilliant blue and sypro ruby.

When separating protein mixtures on 2-D gels for proteomics purposes, fluorescent staining is superior in sensitivity and linear response as compared to Coomassie Brilliant Blue (CBB) and silver staining, respectively. We have compared the quality of mass spectra for proteins obtained from gels stained with CBB and SYPRO Ruby (SR) and found significant differences. These differences can be seen both in inferior signal/noise ratios and number of peptides detected with the fluorescent stain.

Hepatic protein expression of lean mice and obese diabetic mice treated with peroxisome proliferator-activated receptor activators.

The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that modulate lipid and glucose homeostasis. In the clinic, PPARalpha and PPARgamma agonists are used to treat hypertriglyceridemia and insulin resistance of diabetes, respectively. To gain further insight into the molecular mechanisms underlying the therapeutic actions of these drugs, we have by two-dimensional electrophoresis and mass spectrometry performed a comparative analysis of the hepatic protein expression profiles of lean and obese (ob/ob) mice, and obese mice treated with WY14643 (PPARalpha agonist) or rosiglitazone (PPARgamma agonist). We found that livers from obese mice displayed higher levels of enzymes involved in fatty acid oxidation and lipogenesis compared to lean mice and these differences were further amplified by treatment with both PPAR activators. WY14643 normalized the expression levels of several enzymes involved in glycolysis, gluconeogenesis and amino acid metabolism in the obese mice to the levels of lean mice, whereas rosiglitazone partially normalized levels of enzymes involved in amino acid metabolism. In summary, a classical proteomics approach was successfully used to characterize differences at the hepatic proteome level between lean and obese diabetic mice, to map metabolic pathways affected by treatment, and to discriminate between effects caused by treatment with agonists of the closely related PPARalpha and PPARgamma receptors.

Isolation and identification of a novel mitochondrial metalloprotease (PreP) that degrades targeting presequences in plants.

Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form alpha-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F(1)beta subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Two-dimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease in Arabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.