As nanoparticles (NPs) are increasingly used in many applications their safety and efficient applications in nanomedicine have become concerns. Protein coronas on nanomaterials' surfaces can influence how the cell "recognizes" nanoparticles, as well as the in vitro and in vivo NPs' behaviors. The SuperParamagnetic Iron Oxide Nanoparticle (SPION) is one of the most prominent agents because of its superparamagnetic properties, which is useful for separation applications. To mimic surface properties of different types of NPs, a core-shell SPION library was prepared by coating with different surfaces: polyvinyl alcohol polymer (PVA) ( positive, neutral and negative), SiO 2 ( positive and negative), titanium dioxide and metal gold. The SPIONs with different surfaces were incubated at a fixed serum : nanoparticle surface ratio, magnetically trapped and washed. The tightly bound proteins were quantified and identified. The surface charge has a great impact on protein adsorption, especially on PVA and silica where proteins preferred binding to the neutral and positively charged surfaces. The importance of surface material on protein adsorption was also revealed by preferential binding on TiO 2 and gold coated SPION, even negatively charged. There is no correlation between the protein net charge and the nanoparticle surface charge on protein binding, nor direct correlation between the serum proteins' concentration and the proteins detected in the coronas. † Electronic supplementary information (ESI) available. For ESI and crystallographic data in CIF or other electronic format see This journal is

Apart from transporting lipids through the body, the human plasma lipoproteins very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) are also thought to serve as a modality for intra-organismal protein transfer, shipping proteins with important roles in inflammation and thrombosis from the site of synthesis to effector locations. To better understand the role of VLDL and LDL in the transport of proteins, we applied a combination of LTQ ORBITRAP-XL (nLC-MS/MS) with both in-SDS-PAGE gel and in-solution tryptic digestion of pure and defined VLDL and LDL fractions. We identified the presence of 95 VLDL- and 51 LDL-associated proteins including all known apolipoproteins and lipid transport proteins, and intriguingly a set of coagulation proteins, complement system and anti- microbial proteins. Prothrombin, protein S, fibrinogen γ, PLTP, CETP, CD14 and LBP were present on VLDL but not on LDL. Prenylcysteine oxidase 1, dermcidin, cathelicidin antimicrobial peptide, TFPI-1 and fibrinogen α chain were associated with both VLDL and LDL. Apo A-V is only present on VLDL and not on LDL. Collectively, this study provides a wealth of knowledge on the protein constituents of the human plasma lipoprotein system and strongly supports the notion that protein shuttling through this system is involved in the regulation of biological processes. Human diseases related to proteins carried by VLDL and LDL can be divided in three major categories: 1 - dyslipidaemia, 2 - atherosclerosis and vascular disease, and 3 - coagulation disorders.

It is now well recognized that the surfaces of nanoparticles (NPs) are coated with biomolecules (e.g., proteins) in a biological medium. Although extensive reports have been published on the protein corona at the surface of NPs in vitro, there are very few on the in vivo protein corona. The main reason for having very poor information regarding the protein corona in vivo is that separation of NPs from the in vivo environment has not been possible by using available techniques. Knowledge of the in vivo protein corona could lead to better understanding and prediction of the fate of NPs in vivo. Here, by using the unique magnetic properties of superparamagnetic iron oxide NPs (SPIONs), NPs were extracted from rat sera after in vivo interaction with the rat's physiological system. More specifically, the in vivo protein coronas of polyvinyl-alcohol-coated SPIONs with various surface charges are defined. The compositions of the corona at the surface of various SPIONs and their effects on the biodistribution of SPIONs were examined and compared with the corona composition of particles incubated for the same time in rat serum.

IntroductionHodgkin lymphoma (HL) is characterized by a minority of neoplastic cells, the Hodgkin and Reed-Sternberg cells (HRS cells), and an extensive inflammatory background. Many studies have documented that HL is associated with disturbed cytokine production. 1 Cytokine and chemokine production may not only promote growth of HRS cells and help to evade immune surveillance, but also cause the characteristic histology and the clinical symptoms of HL. 2 Cross-talk between HRS cells and surrounding lymphocytes has been studied for many years, and this interaction is regarded to be important for the pathogenesis of HL.The application of proteomics techniques has been proven to be a powerful tool for the identification of new biomarkers involved in pathogenesis of many diseases (reviewed by Hanash 3 ). Some studies on hematologic malignancies using proteomics approaches have been reported, 4,5 but application of proteomics technology to HL is still limited. Fujii et al compared HL cell line proteome to anaplastic large cell lymphoma (ALCL) and non-Hodgkin lymphoma (NHL) using a 2-dimensional difference gel electrophoresis approach of total cell lysates. 6,7 HL expressed higher levels of pyridoxine-5Ј-phosphate oxidase, vinculin, dihydropyrimidinase-related protein-2 and NADHubiquinone oxidoreductase compared with ALCL. In comparison to NHL cell lines, HL cell lines showed higher expression of pyridoxine-5Ј-phosphate oxidase, ␥-enolase, vinculin, vimentin, galectin-1, annexin A5, and protein kinase C substrate. 7 Carvalho et al identified changes in the spectrum using quadrupole-time-of-flight hybrid mass spectrometer in serum of HL patients compared with normal controls, but no further protein identification was performed. 8 Overall, only very limited proteomics data are available for HL.To gain more insight into the proteins secreted by HRS cells that might be involved in the cross-talk with infiltrating lymphocytes or might serve as tumor biomarkers, the secretome of HL cell lines was determined in cell culture supernatant. Protein identification was performed by nanoscale reversed-phase liquid chromatography tandem mass spectrometry (LC-MS/MS) after 1D-SDS-PAGE fractionation. Proteins related to immune response were validated in cell culture supernatant and patient plasma by enzyme-linked immunosorbent assay (ELISA) and in tumor tissues by immunohistochemistry. Methods Cell lines and cultureThe HL cell lines L1236 and KMH2 were established from HL patients with mixed cellularity subtype, L428 from nodular sclerosis subtype and DEV from nodular lymphocyte predominant HL (NLPHL) subtype. They were cultured in RPMI 1640 medium (Lonza Walkersville, Walkersville, MD) supplemented with fetal calf serum (Lonza Walkersville). For analysis of secreted proteins, to avoid the masking effects of high-abundance serum proteins in the culture medium, the cells were washed 3 times with RPMI 1640 medium to deplete all serum proteins, and cultured in RPMI 1640 medium without serum for 24 hours. Under these conditions, cell via...

gamma-Butyrolactone bacterial hormones regulate antibiotic production and morphological differentiation in Streptomyces species. One gamma-butyrolactone, SCB1, has been previously characterized in Streptomyces coelicolor. Here we report the characterization of two additional gamma-butyrolactones, named SCB2 (2-[1'-hydroxyoctyl]-3-hydroxymethylbutanolide) and SCB3 (2-[1'-hydroxy-6'-methyloctyl]-3-hydroxymethylbutanolide), possessing an antibiotic stimulatory activity. To elucidate the specificity determinants of these ligands for the receptor protein, ScbR, 30 chemically synthesized gamma-butyrolactone analogs were tested by utilizing the release of ScbR from DNA upon binding to a gamma-butyrolactone, which can be detected by kanamycin resistance. The butyrolactone detection method developed here revealed that ScbR shows preference toward a ligand possessing a 7-10 carbon C-2 side chain, a C-1'-beta-hydroxyl group, and a C-6'-methyl branch that coincides with SCB3. Moreover, this method was successfully used to screen for potential gamma-butyrolactone producers from commercial-antibiotic-producing Streptomyces.

A Matrix-assisted laser desorption/ionization hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer was employed to acquire neuropeptide mass spectra, directly from neuropeptide secreting tissue deposited on the sample target, in the presence of dihydroxybenzoic acid as matrix. The cockroach corpus cardiacum served as model neuroendocrine tissue. Twelve neuropeptide ion peaks, with mass-to-charge ratio values ranging between 800 and 3,000 Da were selected for tandem mass spectrometry. All peptides below 1,600 Da could be fully sequenced; tandem mass spectrometry analysis of the remaining (three) largest peptides resulted in (limited) sequence tags, which, also due to unavailability of an appropriate neuropeptide structure database, did not allow complete structure elucidation.

The humicolous actinomycete Streptomyces coelicolor routinely adapts to a wide variety of habitats and rapidly changing environments. Upon salt stress, the organism is also known to increase the levels of various compatible solutes. Here we report the results of the first high-resolution metabolomics time series analysis of various strains of S. coelicolor exposed to salt stress: the wild type, mutants with progressive knockouts of the ectoine biosynthesis pathway, and two stress regulator mutants (with disruptions of the sigB and osaB genes). Samples were taken from cultures at 0, 4, 8, and 24 h after salt stress treatment and analyzed by liquid chromatography-mass spectrometry with an LTQ Orbitrap XL mass spectrometer. The results suggest that a large fraction of amino acids is upregulated in response to the salt stress, as are proline/glycine-containing diand tripeptides. Additionally we found that 5-methylthioadenosine, a known inhibitor of polyamine biosynthesis, is downregulated upon salt stress. Strikingly, no major differences between the wild-type cultures and the two stress regulator mutants were found, indicating a considerable robustness of the metabolomic response to salt stress, compared to the more volatile changes in transcript abundance reported earlier.Salt stress conditions are known to cause major changes in the primary and secondary metabolism of bacterial cells (26,34). This is also the case for the Gram-positive soil bacteria of the genus Streptomyces, which are well known for their complex secondary metabolism and production of a wide range of industrially relevant metabolites, including various antibiotics (21). Upon salt stress, these species are known to increase the levels of various compatible solutes, including ectoine, alanine, glutamine, and proline (6,11,16,28). The genome sequence of Streptomyces indicates a largely unexplored capacity for production of secondary metabolites (2), some of which could also be involved in the salt stress response. Here we conducted an untargeted metabolomics analysis of salt-stressed Streptomyces coelicolor, the genome-sequenced model species of the genus (2), to investigate the complexity of the metabolite changes associated with the salt stress response in more detail and to increase our understanding of the mechanisms of osmoadaptation by metabolomic rearrangement. For this purpose, we used a high-resolution LTQ Orbitrap mass spectrometer coupled with hydrophobic-interaction liquid chromatography (HILIC) (23). The accurate mass determination and high resolving power of the Orbitrap (22, 40) have made the analysis of complex metabolite mixtures feasible, making the Orbitrap an attractive option for untargeted metabolomics screens (5). The use of HILIC ensures that the resolving power of the mass spectrometer is further extended by fractionation of the sample, resulting in a less complex mixture being injected in the mass spectrometer at each moment in time. In addition to wild-type S. coelicolor, we studied strains with disruptions in various ste...

Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful and widely applied method for the study of biological systems, biomarker discovery and pharmacological interventions. LC-MS measurements are, however, significantly complicated by several technical challenges, including: (1) ionisation suppression/enhancement, disturbing the correct quantification of analytes, and (2) the detection of large amounts of separate derivative ions, increasing the complexity of the spectra, but not their information content. Here we introduce an experimental and analytical strategy that leads to robust metabolome profiles in the face of these challenges. Our method is based on rigorous filtering of the measured signals based on a series of sample dilutions. Such data sets have the additional characteristic that they allow a more robust assessment of detection signal quality for each metabolite. Using our method, almost 80% of the recorded signals can be discarded as uninformative, while important information is retained. As a consequence, we obtain a broader understanding of the information content of our analyses and a better assessment of the metabolites detected in the analyzed data sets. We illustrate the applicability of this method using standard mixtures, as well as cell extracts from bacterial samples. It is evident that this method can be applied in many types of LC-MS analyses and more specifically in untargeted metabolomics.