Recent developments in pre-treatment and analytical techniques for synthetic polymers by MALDI-TOF mass spectrometry.

A great deal of effort has been expended to develop accurate means of determining the properties of synthetic polymers using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Many studies have focused on the importance of sample pre-treatment to obtain accurate analysis results. This review discusses the history of synthetic polymer characterization and highlights several applications of MALDI-TOF MS that recognize the importance of pre-treatment technologies. The subject area is of significance in the field of analytical chemistry, especially for users of the MALDI technique. Since the 2000s, many such technologies have been developed that feature improved methods and conditions, including solvent-free systems. In addition, the recent diversification of matrix types and the development of carbon-based matrix materials are described herein together with the current status and future directions of MALDI-TOF MS hardware and software development. We provide a summary of processes used for obtaining the best analytical results with synthetic polymeric materials using MALDI-TOF MS.

Reliable Identification of Bacillus cereus Group Species Using Low Mass Biomarkers by MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based pathogen identification relies on the ribosomal protein spectra provided in the proprietary database. Although these mass spectra can discern various pathogens at species level, the spectra-based method still has limitations in identifying closely-related microbial species. In this study, to overcome the limits of the current MALDI-TOF MS identification method using ribosomal protein spectra, we applied MALDI-TOF MS of low-mass profiling to the identification of two genetically related Bacillus species, the food-borne pathogen Bacillus cereus, and the insect pathogen Bacillus thuringiensis. The mass spectra of small molecules from 17 type strains of two bacilli were compared to the morphological, biochemical, and genetic identification methods of pathogens. The specific mass peaks in the low-mass range (m/z 500- 3,000) successfully identified various closely-related strains belonging to these two reference species. The intensity profiles of the MALDI-TOF mass spectra clearly revealed the differences between the two genetically-related species at strain level. We suggest that small molecules with low molecular weight, 714.2 and 906.5 m/z can be potential mass biomarkers used for reliable identification of B. cereus and B. thuringiensis.

Genome Sequence of an Endophytic Fungus, Fusarium solani JS-169, Which Has Antifungal Activity.

An endophytic fungus, Fusarium solani strain JS-169, isolated from a mulberry twig, showed considerable antifungal activity. Here, we report the draft genome sequence of this strain. The assembly comprises 17 scaffolds, with an N50 value of 4.93 Mb. The assembled genome was 45,813,297 bp in length, with a G+C content of 49.91%.

Mass spectrometric screening of ovarian cancer with serum glycans.

Changes of glycosylation pattern in serum proteins have been linked to various diseases including cancer, suggesting possible development of novel biomarkers based on the glycomic analysis. In this study, N-linked glycans from human serum were quantitatively profiled by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and compared between healthy controls and ovarian cancer patients. A training set consisting of 40 healthy controls and 40 ovarian cancer cases demonstrated an inverse correlation between P value of ANOVA and area under the curve (AUC) of each candidate biomarker peak from MALDI-TOF MS, providing standards for the classification. A multibiomarker panel composed of 15 MALDI-TOF MS peaks resulted in AUC of 0.89, 80~90% sensitivity, and 70~83% specificity in the training set. The performance of the biomarker panel was validated in a separate blind test set composed of 23 healthy controls and 37 ovarian cancer patients, leading to 81~84% sensitivity and 83% specificity with cut-off values determined by the training set. Sensitivity of CA-125, the most widely used ovarian cancer marker, was 74% in the training set and 78% in the test set, respectively. These results indicate that MALDI-TOF MS-mediated serum N-glycan analysis could provide critical information for the screening of ovarian cancer.

In situ identification and localization of IGHA2 in the breast tumor microenvironment by mass spectrometry.

Modifications in the tumor microenvironment (TME) play a major role in the establishment, progression, and metastasis of cancer. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique that enables the simultaneous identification and localization of biological compounds within tissues. To detect markers of early TME remodeling in invasive breast cancer, we used MALDI-MSI to compare the molecular profiles of tissues from the breast cancer interface zone, tumor zone, and normal-tissue zone. Using direct-tissue MALDI tandem mass spectrometry (MS/MS), we identified immunoglobulin heavy constant alpha 2 (IGHA2) as a new, zone-specific protein in the breast TME. The zone-specific expression of IGHA2 was verified by immunoblotting and immunohistochemical analysis. IGHA2 expression was consistently positive in tumor cells that were metastatic to regional nodes, with intense expression along the cytoplasmic borders. As a factor related to an increased percentage of nodes with tumor metastasis, IGHA2 expression was upregulated 3.745-fold in cases with an increased number of cancerous nodes (p = 0.0468). Our results provide the first evidence of IGHA2 as a marker of the early process of TME remodeling in invasive breast cancer. Furthermore, IGHA2 may be a novel marker for regional metastases in the lymph nodes of patients with breast cancer.

Analysis of oligosaccharides in beer using MALDI-TOF-MS.

Oligosaccharides in four different brands of beer (Cass, Hite, Budweiser, Miller) were systematically analysed with three different dihydroxybenzoic acid (DHB) isomer matrices (2,4-DHB, 2,5-DHB, and 2,6-DHB) using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Different experimental conditions, such as dilution (up to 1000-fold) and cationisation agents (sodium chloride or sodium trifluoroacetate) were analysed. No ionised peaks of oligosaccharides were observed with 2,4-DHB matrix. 2,6-DHB was more effective than 2,5-DHB in most of the investigated concentration ranges. 2,6-DHB with 4-fold dilution was the most effective. In certain cases, a cationisation agent was necessary to detect the signals of the oligosaccharides, and sodium chloride provided greater ionisation than sodium trifluoroacetate.

High-throughput small molecule identification using MALDI-TOF and a nanolayered substrate.

Encoderless combinatorial chemistry requires high-throughput product identification without the use of chemical or other tags. We developed a novel nanolayered substrate plate and combined it with a microarraying robot, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, and custom software to produce a high-throughput small molecule identification system. To optimize system performance, we spotted 5 different chemical entities, spanning a m/z range of 195 to 1338, in 20,304 spots for a total of 101,520 molecules. The initial spot identification rate was 99.85% (20,273 spots), and after a proofreading algorithm was added, 100% of 20,304 spots and 101,520 molecules were identified. An internal recalibration algorithm also significantly improved mass accuracy to as low as 45 ppm. Using this optimized system, 47 different chemical entities, spanning a m/z range of 138 to 1,592, were spotted over 5,076 spots and could be identified with 100% accuracy. Our study lays the foundation for improved encoderless combinatorial chemistry.

Enrichment of phosphopeptides using bare magnetic particles.

Magnetic iron(II, III) oxide (magnetite, Fe(3)O(4)) nanoparticles were used to selectively enrich phosphopeptides from tryptic digests of bovine beta-casein and from tryptic digest mixtures containing bovine beta-casein, cytochrome c, bovine serum albumin, and horse heart myoglobin. The magnetic property of the particles permits an easy and speedy enrichment process. No enrichment of phosphopeptides was observed from ferric magnetic iron(III) oxide (Fe(2)O(3)) nanoparticles. These data collectively demonstrate that the enrichment of phosphopeptides using magnetic iron(II, III) oxide nanoparticles is a practical method for the selective analysis of phosphopeptides and could be helpful in isolating and analyzing phosphorylated peptides from complex biological samples.

Molecular mechanism of bis(maltolato)oxovanadium(IV)-induced insulin signaling in 3T3-L1 and IM9 cells: impact of dexamethasone.

The organovanadium compound bis(maltolato)oxovanadium(IV) (BMOV) enhanced the tyr-phosphorylation of major upstream insulin signaling proteins including the vital site-specific phosphorylation of insulin receptor beta (IRbeta) in IM9 and 3T3-L1 cells in dose- and time-dependent manners more efficiently than insulin. Nevertheless, insulin in general had a synergistic impact on those phosphorylations in both cell lines, while its presence was obligatory to induce Tyr(972)-phosphorylation of IRbeta in IM9 cells at 18-h treatment with BMOV. However, prolonged exposure of cells to BMOV caused depletion in IR level and using IM9 cells we found that this event was counteracted by insulin, where monensin, a monocarboxylic acid ionophore made an additive impact, suggesting that a novel mechanism is being involved in the recycling of internalized IR in BMOV-treated cells. On the other hand, dexamethasone elevated the IR level in both cell lines. However, no correlation was found between the cellular content and the degree of phosphorylation of IRbeta in cells receiving combined treatment of BMOV, and dexamethasone with short insulin post-exposure. BMOV also induced the phosphorylation of Thr(308) and Ser(473) of Akt in both cell lines receiving insulin post-treatment, while dexamethasone decreased those phosphorylations. However, this activation/deactivation of Akt did not correlate with the phosphorylation status of Ser(9) and Ser(259) of glycogen synthase kinase (GSK)-3beta and Raf respectively. Taken together, it is conceivable that BMOV and/or dexamethasone modulate insulin signaling by acting differentially on the components of the insulin signaling network. We also consider that the observed dexamethasone-mediated modulation of insulin receptor kinase in BMOV-treated 3T3-L1 cells probably occurs through the activation/deactivation of some mechanism which needs further studies for proper characterization.

Vanadium and insulin increase adiponectin production in 3T3-L1 adipocytes.

Both adiponectin, an adipokine secreted by adipocytes, and vanadium compounds, have been extensively shown to enhance insulin sensitivity in vivo and in vitro. In this study we examined whether insulin and vanadyl sulfate (VS) affected adiponectin release and cell content from 3T3-L1 adipocytes, and whether they acted through a similar signaling pathway. Adiponectin cell content, but not release, consistently increased in cells treated with insulin (100 nM) and VS (10 and 50 microM) after 24 h. On the other hand, VS-induced adiponectin release only occurred after 4 days of incubation. The protein kinase B (PKB) inhibitor, NL-71-101, decreased both insulin and VS-induced adiponectin cell content, while neither wortmannin nor LY 294002, inhibitors of phosphatidylinositol 3-kinase (PI3-K), attenuated insulin or VS-induced adiponectin cell content. Furthermore, VS-induced adiponectin accumulation occurred in the presence of AGL2263, an insulin receptor (IR) inhibitor. These studies provide the first evidence that vanadium could exert its insulin sensitizing effects through the stimulation of adiponectin through a PKB-dependent transduction pathway.

Analysis of glycated insulin by MALDI-TOF mass spectrometry.

Non-enzymatic glycation of protein is mediated via an interaction between the aldehyde group of a reducing sugar and available alpha- or epsilon-amino moieties of the protein. The above event can alter the biological activity of the protein and therefore, it is of particular interest to monitor the glycation of proteins having important functional roles in metabolism. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) has been used to determine the non-enzymatic glycation of bovine insulin. The degree of insulin glycation was increased in both concentration- and time-dependent manner in relation to exposure to glucose, and the event was more pronounced for monoglycation reaction than that noticed for the diglycation of the hormone. Enzymatic digestion of insulin preparations with endoproteinase Glu C has revealed that each of the B 1-13 and B 22-30 peptide fragments of glycated insulin contains a site of binding of a single glucose molecule. Finally, attempt has been made in order to increase the sensitivity of the glycation assay through efficient enrichment of the glycated insulin on magnetic beads containing immobilized 3-aminophenylboronic acid (APBA) on their surface.

Immobilization of aminophenylboronic acid on magnetic beads for the direct determination of glycoproteins by matrix assisted laser desorption ionization mass spectrometry.

Aminophenylboronic acid (APBA) has been immobilized on magnetic beads for the direct determination of glycoprotein by matrix assisted laser desorption/ionizaton time of flight mass spectrometry (MALDI-TOF-MS). An APBA layer was formed on the surface of carboxylic acid terminated magnetic beads by coupling with carbodiimide and subsequently reacted with an N-hydroxysuccinimide moiety. The immobilized APBA was identified by MALDI-TOF-MS without a matrix. Glycoproteins, such as HbA1c, fibrinogen, or RNase B were separated and desalted using APBA magnetic beads by simply washing the magnetic beads and then separating them by external magnet. Proteins can be identified by direct determination of proteins on beads on MALDI plate and confirmed again by peptide mass finger printing after digestion of proteins on magnetic beads by trypsin. Fluorescence image with a FITC tagging protein using confocal laser microscopy showed the difference of immobilization efficiency between glycoproteins and nonglycoproteins. The methods developed within this work allow the simple treatment and enrichment of glycoproteins as well as direct determination of proteins on beads by MALDI-TOF-MS.

Low-pressure ICP-AES with electrothermal vaporizer and a pneumatic nebulizer for aqueous sample introduction.

A typical electrothermal vaporization (ETV) using a tantalum was built for low-pressure ICP-AES. The analytical performance of the ETV was tested and compared with that of a PFA pneumatic nebulizer with a double membrane desolvator (DMD). The limits of detection of the ETV were obtained in the range of 3.4 ng to 758 ng for Zn, Cu, Co, Fe, and Mg, while those of the PFA nebulizer were in the range of 53 ppb to 286 ppb. A relative standard deviation (RSD) of 4.3 - 8.5% for ETV was obtained, while 2.15 - 6.84% RSD was found for DMD.

Development of an automated protein-tyrosine phosphatase 1B inhibition assay and the screening of putative insulin-enhancing vanadium(IV) and zinc(II) complexes.

The inhibition of protein-tyrosine phosphatase 1B (PTP1B) is a potential target for treatment of type 2 diabetes. Vanadium and zinc metal coordinated complexes have insulin-enhancing activities, and while vanadium compounds inhibit PTP1B, little is known on the mode of action of zinc compounds. In this study we developed an automated PTP1B inhibition assay that allows for a rapid assessment of the PTP1B inhibition strength of candidate compounds. Synthetic vanadium(IV) and zinc(II) complexes were evaluated: IC50 values for vanadium complexes ranged from 0.06 to 0.8 microM whereas for zinc compounds, values were above 10 microM. Vanadium sulfate, a non-conjugated inorganic salt, had stronger inhibition activity than any of the conjugated metal complexes.

Purification and characterization of metallothionein-like cadmium binding protein from Asian periwinkle Littorina brevicula.

Although mussels and oysters in the ocean are known to act as bioconcentrators for contaminants such as heavy metals, their ability to survive in heavily polluted water is relatively limited. The Asian periwinkle, Littorina brevicula, is one species that can accumulate a variety of environmental heavy metals, and the expression of its metal binding protein (MBP) is induced by cadmium. To better characterize this protein and its detoxification mechanism against cadmium, the present work examined the induction of a cadmium binding protein (Cd-BP) in Littorina brevicula exposed to 400 microg/l CdCl(2) for 30 days. The induced Cd-BP was purified by chromatography from the supernatants of homogenized organs (digestive gland, gonad, gill and kidney). This Cd-BP was found to consist of 103 amino acids, was rich in Cys (21 residues), and partial C-terminal sequence obtained by MALDI-TOF MS analysis revealed a Cys-XXX-Cys motif, which resembles a typical feature of mollusc metallothionein (MT). The Cd-BP molecular weight of 9.8 kDa is a little larger than that of other MTs.