The effect of starvation stress on Lactobacillus brevis L62 protein profile determined by de novo sequencing in positive and negative mass spectrometry ion mode.

RATIONALE: We describe a novel negative chemically activated fragmentation/positive chemically activated fragmentation (CAF-/CAF+) technique for protein identification. The technique was used to investigate Lactobacillus brevis adaptation to nutrient deprivation. METHODS: The CAF-/CAF+ method enables de novo sequencing of derivate peptides with negative and positive ion mode matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS). Peptide sequences obtained from MS/MS spectra were matched against the National Center for Biotechnology Information (NCBI) non-redundant (nr) database and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome. This improved protein identification method highlighted 36 differentially expressed proteins in the proteome of L. brevis after 75 days of starvation. RESULTS: The results revealed the key differences in the metabolic pathways that are responsible for the survival of L. brevis in a hostile environment. Proteomics analysis demonstrated that numerous proteins engaged in glucose and amino-acid catabolizing pathways, glycerolipid metabolizing pathways, and stress-response mechanisms are differentially expressed after long-term starvation. Amino acid and proteomics analysis indicated that starved L. brevis metabolized arginine, glycine, and histidine from dead cells as alternative nutrient sources. The production of lactic acid also varied between the parent cells and the starved cells. CONCLUSIONS: Differentially expressed proteins identified exclusively by peptide sequence reading provided promising results for CAF-/CAF+ implementation in a standard proteomics workflow (e.g., biomarker and mutation discovery and biotyping). The practical performance of a reliable de novo sequencing technique in routine proteomics analysis is emphasized in this article.

A product of ozonolysis of cholesterol alters the biophysical properties of phosphatidylethanolamine membranes.

There is evidence that some products of the reaction of ozone with cholesterol contribute to atherosclerosis. One of these compounds is 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al. We have synthesized this compound and have demonstrated that it reacts with phosphatidylethanolamine to form a Schiff base. The 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al also affects the physical properties of phosphatidylethanolamines. We show by both DSC and X-ray diffraction that it increases the negative curvature of the membrane. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al causes the lamellar phase to become disorganized, resulting in the loss of lamellar periodicity. The chemical and physical interactions of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al with phosphatidylethanolamines may contribute to damaging effects of this lipid on cell membranes, resulting in pathology.