Characterization of particulate organic matters in the water column of the South China Sea using a shotgun proteomic approach

We characterized particulate organic matter (POM) collected from both the surface (41 m and 200 m) and mesopelagic layers (500 m and 1000 m) in the western South China Sea. By using a shotgun proteomic approach, a total of 3035 proteins matching one or more peptides were detected from four POM samples, 505 of which were identified as high-confidence proteins matching two or more peptides. Cyanobacteria was the largest contributor throughout the water column, while crustaceans and dinophytes were the two major groups contributing to the particulate proteins in the POM collected from 200 m. Subcellular locations and biological processes of particulate proteins varied significantly between the 41-m and 200-m layers: photosynthesis-associated proteins were highly abundant in the 41-m layer while tubulins and actins accumulated in the midwaters, especially at the 200-m layer. Porins, adenosine triphosphate synthases, nutrient transporters, molecular chaperones, and ectoenzymes were frequently detected in the POM samples and presented different distribution patterns within the water column, revealing complex biological processes at the different water layers and/or during the sinking of POM. The sources of surface and midwater particulate proteins are different, and the cellular metabolism, generation of energy, and transport processes in POM are attenuated rapidly down ocean water column. Zooplankton fecal pellet packages and membrane encapsulation might play important roles in protecting particulate proteins from degradation.Ministry of Science and Technology [2009CB421203]; National Natural Science Foundation of China [40821063, 40776068, 40876059]; Program for New Century Excellent Talents in University of Chin

Homology-Driven Proteomics of Dinoflagellates with Unsequenced Genomes Using MALDI-TOF/TOF and Automated De Novo Sequencing

This study developed a multilayered, gel-based, and underivatized strategy for de novo protein sequence analysis of unsequenced dinoflagellates using a MALDI-TOF/TOF mass spectrometer with the assistance of DeNovo Explorer software. MASCOT was applied as the first layer screen to identify either known or unknown proteins sharing identical peptides presented in a database. Once the confident identifications were removed after searching against the NCBInr database, the remainder was searched against the dinoflagellate expressed sequence tag database. In the last layer, those borderline and nonconfident hits were further subjected to de novo interpretation using DeNovo Explorer software. The de novo sequences passing a reliability filter were subsequently submitted to nonredundant MS-BLAST search. Using this layer identification method, 216 protein spots representing 158 unique proteins out of 220 selected protein spots from Alexandrium tamarense, a dinoflagellate with unsequenced genome, were confidently or tentatively identified by database searching. These proteins were involved in various intracellular physiological activities. This study is the first effort to develop a completely automated approach to identify proteins from unsequenced dinoflagellate databases and establishes a preliminary protein database for various physiological studies of dinoflagellates in the future

Identification and Characterization of Cell Wall Proteins of a Toxic Dinoflagellate Alexandrium catenella

The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated from A. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database using de novo sequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions

Metaproteomic characterization of dissolved organic matter in the water column of the South China Sea

Ministry of Science and Technology [2009CB421203]; National Natural Science Foundation of China [40821063, 40776068, 40876059]; University of ChinaWe characterized dissolved organic matter (DOM; < 0.7 mu m in size) collected from the surface (10-m and 75-m) and bathypelagic (3000-m) layers in the South China Sea using the shotgun proteomic approach. A total of 182 proteins matched by 286 unique peptides were identified from three DOM samples. The number of proteins in the large DOM (LDOM; 0.2-0.7-mu m fraction) was significantly greater than that in the small DOM (SDOM; 5-kD-0.2-mu m fraction). There was no remarkable difference in the number of proteins between the surface and bathypelagic SDOMs. The sources of dissolved proteins were diverse in surface DOM, including various bacterial and phytoplankton groups as well as Oomycetes, whereas the Archaea, Proteobacteria, and some phytoplankton groups were the major contributors to bathypelagic DOM. Proteins involved in cytoskeleton, energy production and conversion, posttranslational modification, protein turnover, and chaperones presented high abundance in surface LDOM, whereas proteins involved in translation, ribosomal structure, and biogenesis were more abundant in bathypelagic LDOM. Proteins involved in transport and metabolism, cell wall or membrane or envelope biogenesis, and photosynthesis were abundant in the 75-m LDOM. A urea ABC transporter assigned to amino acid transport and metabolism was the most abundant protein in the 10-m SDOM, whereas methylenetetrahydromethanopterin reductase involved in energy production and conversion dominated the protein profiles in the 75- and 3000-m SDOMs. The dissolved proteins in the water column are diverse and dynamic, with each layer characterized by unique proteins, and only a very minor amount of proteins from the surface are protected and transferred to the deep sea

Identification and Characterization of Cell Wall Proteins of a Toxic Dinoflagellate Alexandrium catenella Using 2-D DIGE and MALDI TOF-TOF Mass Spectrometry

Specialized Research Fund for the Doctoral Program of Higher Education [20070384014]; National Natural Science Foundation of China [40776068, 40876059]; Ministry of Science and Technology of the People's Republic of China [2010CB428703]; Excellent Group;The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated from A. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database using de novo sequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions

Evaluation of Chitosan Derivative Microparticles Encapsulating Superparamagnetic Iron Oxide and Doxorubicin as a pH-Sensitive Delivery Carrier in Hepatic Carcinoma Treatment: An in vitro Comparison Study

We developed a novel, pH-sensitive drug delivery microparticle based on N-palmitoyl chitosan (NPCS) to transport the superparamagnetic iron oxide (SPIO) and anticancer drug doxorubicin (DOX). The characteristics of NPCS were characterized through nuclear magnetic resonance. Our results based on testing of volume swelling in multiple pH aqueous solutions revealed that the modified chitosan had a pH-sensitive property. The morphology and size of the DOX-SPIO/NPCS microparticles were investigated using transmission electron microscopy and scanning electron microscopy. The statistical result of microparticles had diameter of 185 ± 87 nm. Surface chemical moieties of DOX-SPIO/NPCS microparticles were confirmed using attenuated total reflection Fourier transform infrared spectroscopy and indicated the existence of mostly hydrophilic groups such as -OH, -C=O, and -C-O-C-. Transmission electron microscopy revealed the dark contrast of SPIO dots encapsulated in the NPCS matrix. Nuclear magnetic resonance T2-weighted magnetic resonance imaging confirmed that the produced DOX-SPIO/NPCS microparticles still exhibited T2 relaxation durations as short as 37.68 ± 8.69 ms (under administration of 2.5 μg/mL), which is comparable to the clinically required dosage. In the drug release profile, the DOX-SPIO/NPCS drug delivery microparticle was accelerated in an acidic environment (pH 6.5) compared with that in a basic environment. Microparticles in a cytotoxicity assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay) revealed that DOX-SPIO/NPCS microparticles had better antitumor ability than did free-form of DOX. Additionally, microparticles loaded with 0.5–5 μg/mL DOX in an acidic environment (pH 6.5) demonstrated higher efficacy against Hep G2 cell growth, possibly because of the swelling effect of NPCS, resulting in volume expansion and easy drug release. Accordingly, these large DOX-SPIO/NPCS microparticles showed potential for application as a pH-sensitive drug delivery system and as chemoembolization particles for hepatic carcinoma therapy