Determination of isotopic labeling of proteins by precursor ion scanning liquid chromatography/tandem mass spectrometry of derivatized amino acids applied to nuclear magnetic resonance studies.

RATIONALE: A method has been developed for the quantitation of isotopic labeling of proteins using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the application of protein nuclear magnetic resonance (NMR) studies. NMR relies on specific isotopic nuclei, such as (13)C and (15)N, for detection and, therefore, isotopic labeling is an important sample preparation step prior to in-depth structural characterization of proteins. The goal of this study was to develop a robust quantitative assay for assessing isotopic labeling in proteins while retaining information on the extent of labeling for individual amino acids. METHODS: Complete digestion of proteins by acid hydrolysis was followed by derivatization of free amino acids with 6-aminoquinolyl N-hydroxysuccinimidyl carbamate (AQC) forming derivatives having identical MS/MS fragmentation behavior. Precursor ion scanning on a hybrid quadrupole-linear ion trap platform was used for amino acid analysis and determining isotopic labeling of proteins. RESULTS: Using a set of isotope-labeled amino acid standards mixed with their unlabeled counterparts, the method was validated for accurately measuring % isotopic contribution. We then applied the method for determining the (13)C isotopic content of algal proteins during a feeding study using (13)C(6)-glucose- or (13)C-bicarbonate-supplemented culture media as well as the level of labeling in mussel byssal threads obtained after feeding with labeled algae. CONCLUSIONS: This method is ideally suited for assessing the extent of protein labeling prior to NMR studies, where the isotopic labeling is a determining factor in the quality of resulting protein spectra, and can be applied to a multitude of different biological samples.

Stability of carotenoids in Scenedesmus almeriensis biomass and extracts under various storage conditions.

Scenedesmus almeriensis biomass is a source of carotenoids, particularly lutein, and is considered to be promising as an alternative source to marigold. One key question concerning alternative sources of lutein is the loss of carotenoids that takes place between harvesting and processing, which in the case of marigold is frequently up to 50%. The work described here involved a study into the stability of the main carotenoids (lutein, violaxanthin, and beta-carotene), as well as other components, under different storage conditions. The experiments were carried out with biomass in three forms: frozen, freeze-dried, and spray-dried. The stability of extracts of Scenedesmus biomass in acetone and olive oil was also studied. The results show that the most important factor in retaining carotenoids is a low temperature. At -18 degrees C the loss of carotenoids was negligible after the storage period, regardless of the biomass form used (frozen, freeze-dried, or spray-dried). On the other hand, the carotenoid content and fatty acid profile was increasingly affected with increasing temperature. However, the protein content is unaffected by storage conditions.

Influence of phosphorus on Microcystis growth and the changes of other environmental factors.

The growth processes of Microcystis aeruginosa (FACHB-41) in simulated Taihu Lake water with different phosphorus concentrations were investigated using laboratory microcosms. The algal biomass increased with the increase of phosphorus concentration when it was lower than 0.445 mg/L, while the dissolved oxygen (DO) and pH increased, dissolved inorganic nitrogen(DIN) and light intensity underwater (I) decreased. Responding to the changes of the "environmental factors", the cellular carbohydrate and its ratio to cellular protein decreased generally as phosphorus increased. However, when phosphorus concentration was higher than 1.645 mg/L, the biomass, the "environmental factors", the cellular carbohydrate and its ratio to cellular protein did not change likewise. Since the environmental factors and the physiological and biochemical responses are important factors, the change of environmental factors and cell physiology and biochemistry induced by phosphorus may become the key factors that steer the growth and dominance of Microcystis under certain conditions. To sum up, phosphorus not only stimulate the growth of Microcystis directly by supplying nutrient element, but also has complex interactions with other "environmental factors" and play important roles in the growth processes of Microcystis.