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Acrylamide is a group 2A carcinogen and potential endocrine disruptor that can enter the ecosystem by various routes and has recently become a dangerous pollutant. This widely used chemical can enter the human body via air inhalation, food or water consumption, or skin contact. In this study, we developed a peptide probe for the detection of acrylamide by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) after its micro-tagging with a peptide. Direct detection of acrylamide by MALDI-TOF MS is not feasible due to its poor ionization in the MALDI interface, which hinders its analysis by the technique. After microwave irradiation for 2 min, the formed acrylamide-peptide derivative was detected easily by MALDI-TOF MS without the need for extraction procedures. The procedure does not involve organic solvents and a water-soluble peptide that allows detection of acrylamide in small sample volumes with a limit of detection (LOD) of 0.05 ng/μL. The relative standard deviation (RSD) and relative error (RE) of the measurements were

Thrombospondin-1 (TSP1) is involved in corneal wound healing caused by chemical injury. Herein, we examined the effects of TSP1 on hypoxia-induced damages and wound-healing activity in human corneal epithelial (HCE) cells. Exosomal protein expression was determined using liquid chromatography-tandem mass spectrometry, and HCE cell migration and motility were examined through wound-healing assay and time-lapse microscopy. Reestablishment of cell junctions by TSP1 was assessed through confocal microscopy and 3D image reconstruction. Our results show that CoCl2 -induced hypoxia promoted HCE cell death by paraptosis. TSP1 protected these cells against paraptosis by attenuating mitochondrial membrane potential depletion, swelling and dilation of endoplasmic reticulum and mitochondria, and mitochondrial fission. Exosomes isolated from HCE cells treated with TSP1 contained wound healing-associated proteins that were taken up by HCE cells to promote tissue remodeling and repair. TSP1 protected HCE cells against hypoxia-induced damages and inhibited paraptosis progression by promoting cell migration, cell-cell adhesion, and extracellular matrix remodeling. These findings indicate that TSP1 ameliorates hypoxia-induced paraptosis in HCE cells and promotes wound healing and remodeling by regulating exosomal protein expression. TSP1 may, therefore, play important roles in the treatment of hypoxia-associated corneal diseases.

Context Small extracellular vesicles (sEVs) have emerged as modulators of the disease microenvironment, thereby supporting disease progression. However, the potential role of EVs and their content to the pathophysiology of endometriosis remain unclear. Objective This work aimed to investigate whether the EVs from eutopic (Eu) and ectopic (Ec) endometrial stromal cells (ESCs) differ with respect to protein composition and role in endometriosis. Methods Human Eu and Ec endometrium–derived ESCs were isolated from samples of the same patients (n = 3). sEVs were isolated from ESCs via ultracentrifugation; these sEVs were characterized by Western blotting, transmission electron microscopy, and nanoparticle tracking analysis and analyzed using mass spectrometry. The potential role of EcESCs-derived sEVs (EcESCs-sEVs) in endometriosis was explored by assaying their effects on cell viability/proliferation, migration, and angiogenesis. Results In total, 105 ESCs-sEV–associated proteins were identified from EcESCs-sEVs and EuESCs-sEVs by mass spectrometry analysis. The protein content differed between EcESCs-sEVs and EuESCs-sEVs, with annexin A2 (ANXA2) being the most prominent difference—present in EcESCs-sEVs but not EuESCs-sEVs. We also found that sEVs-ANXA2 regulates the motility, proliferation, and angiogenesis of ESCs via the extracellularly regulated kinase (ERK)/STAT3 pathway. Notably, treatment of ESCs with sEVs-ANXA2 resulted in increased proliferation and motility, suggesting that sEVs-ANXA2 may be involved in regulating endometriosis. Our data suggest that EcESCs-sEVs-ANXA2 regulates the motility and the angiogenic potential of ESCs, implying a role for sEVs-ANXA2 in the pathogenesis of endometriosis. Conclusion The study of sEVs-ANXA2 from Ec endometriotic cells uncovers a new mechanism of endometriosis progression and will inform the development of novel therapeutic strategies.

The alternative transcription factor σB in Bacillus cereus governs the transcription of a number of genes that confer protection against general stress. This transcription factor is regulated by protein-protein interactions among RsbV, RsbW, σB, RsbY, RsbM and RsbK, all encoded in the sigB cluster. Among these regulatory proteins, RsbV, RsbW and σB comprise a partner-switching mechanism. Under normal conditions, σB remains inactive by associating with anti-sigma factor RsbW, which prevents σB from binding to the core RNA polymerase. During environmental stress, RsbK activates RsbY to hydrolyze phosphorylated RsbV, and the dephosphorylated RsbV then sequesters RsbW to liberate σB from RsbW. Although the σB partner-switching module is thought to be the core mechanism for σB regulation, the actual protein-protein interactions among these three proteins in the cell remain to be investigated. In the current study, we show that RsbW and RsbV form a long-lived complex under transient stress treatment, resulting in high persistent expression of RsbV, RsbW and σB from mid-log phase to stationary phase. Full sequestration of RsbW by excess RsbV and increased RsbW:RsbV complex stability afforded by cellular ADP contribute to the prolonged activation of σB. Interestingly, the high expression levels of RsbV, RsbW and σB were dramatically decreased beginning from the transition stage to the stationary phase. Thus, protein interactions among σB partner-switching components are required for the continued induction of σB during environmental stress in the log phase and significant down-regulation of σB is observed in the stationary phase. Our data show that σB is temporally regulated in B. cereus.

Background Skin cancer is an important environmentally-related health issue. Although sun exposure is closely associated with increasing environmental heat, the effects of environmental heat on the skin, especially in the context of photocarcinogenesis, has not been carefully examined. Objectives This study aimed to explore the effects and interactions of UVB radiation and environmental heat on photocarcinogenesis of the skin using cell and animal models. Methods Cultured keratinocytes and hairless mice were exposed to different treatment conditions including UVB radiation and environmental heat. The effects of treatment on keratinocyte and mice skin were evaluated at indicated time points. Results UVB induced DNA damage was significantly lower in keratinocytes that were pretreated in an environment with slightly elevated temperature followed by UVB treatment (Heat-UVB) as compared to UVB and UVB radiation followed by exposure to equivalent increase in environmental heat (UVB-Heat) groups. Similar phenomenon was observed in terms of keratinocyte viability. In the animal model, it was found that Heat-UVB treated mice showed delayed and reduced tumor formation as compared to the UVB and UVB-Heat treated groups. Quantum simulation analyses demonstrated that the energy required for CPD formation at environment with higher temperature required considerable higher energy as compared to CPD formation at lower temperature. Conclusion Taken together, our results demonstrated that with equivalent UVB exposure, higher temperature environment may protect cells against subsequent UVB-induced DNA damages.

Reactive oxygen species (ROS) can induce oxidative stress and are associated with cell death and chronic diseases in organisms. In the treatment of disease, drugs that induce ROS are associated with many side effects and unpleasant symptoms. Therefore, during the assessment of new drugs and candidate compounds, ROS generation is an issue of concern, because ROS can modify proteins, lipids, and nucleic acids within organisms and alter their biological functions. In this work, we designed a peptide-based probe for the rapid (

Based on the oxidative stress theory, aging derives from the accumulation of oxidized proteins induced by reactive oxygen species (ROS) in the cytoplasm. Hydrogen peroxide (H2O2) elicits ROS that induces skin aging through oxidation of proteins, forming disulfide bridges with cysteine or methionine sulfhydryl groups. Decreased Ca2+ signaling is observed in aged cells, probably secondary to the formation of disulfide bonds among Ca2+ signaling-related proteins. Skin aging processes are modeled by treating keratinocytes with H2O2. In the present study, H2O2 dose-dependently impaired the adenosine triphosphate (ATP)-induced Ca2+ response, which was partially protected via co-treatment with β-mercaptoethanol, resulting in reduced disulfide bond formation in inositol 1, 4, 5-trisphosphate receptors (IP3Rs). Molecular hydrogen (H2) was found to be more effectively protected H2O2-induced IP3R1 dysfunction by reducing disulfide bonds, rather than quenching ROS. In conclusion, skin aging processes may involve ROS-induced protein dysfunction due to disulfide bond formation, and H2 can protect oxidation of this process.

Valproic acid (VA) is the drug of choice for managing epilepsy syndrome and is an effective treatment for various seizures. In recent years, new applications of VA have been developed for clinical treatment of other diseases. In the degradation pathway, cytochrome P450 participates in the biotransformation of VA to other metabolites. The resulting compounds then contribute to apoptosis induced by oxidative stress. Hence, clinical applications of VA medication should be selected carefully to avoid potentially severe side effects such as hepatotoxicity, hypersensitivity syndrome reactions, neurological toxicity and teratogenicity. This work established a simple method of using a microwave assisted derivatization procedure for improved detection and monitoring of VA derivatives by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The calibration curve was 1–100 μM for VA analysis, and good linearity was obtained. In intra- and inter-day analyses, all relative standard deviation (RSD) and relative error (RE) values were below 7.5%.

In the clinic, ethosuximide is commonly used to treat generalized absence seizures but has recently been repurposed for other diseases. Because of adverse effects and drug interactions, high-throughput therapeutic drug monitoring of ethosuximide is necessary. Microextraction is a simple, effective, rapid, and low consumption of organic solvents method for sample preparation. In this study, microderivatization-increased detection (MDID)-combined microextraction was used to detect ethosuximide by mass spectrometry. Ethosuximide is a difficult to retain and ionize compound in the C18 nano-flow column and ionization interface, respectively. Hence, we developed a fast method for detecting ethosuximide in human plasma by using the MDID strategy (within 2 min). Chemical microderivatization parameters were studied and optimized to increase the sensitivity of ethosuximide detection at trace levels. The linear range for the analysis of ethosuximide in 10 μL plasma was 5–500 μg/mL with a coefficient of determination (r2) ≥ 0.995. The precision and accuracy of intraday and interday analyses of ethosuximide were below 13.0%. Furthermore, modifications of major proteins in plasma and blood cells, induced by ethosuximide, were identified. The proposed method effectively utilizes microliter samples to detect drug plasma concentrations under suitable microextraction procedures toward the eco-friendly goal of low consumption of organic solvents.

Although most reef-building corals live near the upper threshold of their thermotolerance, some scleractinians are resilient to temperature increases. For instance, Pocillopora acuta specimens from an upwelling habitat in Southern Taiwan survived a 9-month experimental exposure to 30°C, a temperature hypothesized to induce stress. To gain a greater understanding of the molecular pathways underlying such high-temperature acclimation, the protein profiles of experimental controls incubated at 27°C were compared to those of conspecific P. acuta specimens exposed to 30°C for two, four, or eight weeks, and differentially expressed proteins (DEPs) were removed from the protein gels and sequenced with mass spectrometry. Sixty unique DEPs were uncovered across both eukaryotic compartments of the P. acuta-dinoflagellate (genus Symbiodinium) mutualism, and Symbiodinium were more likely to up-regulate protein expression in response to high temperature exposure than the coral hosts in which they resided at the 2-week sampling time. Furthermore, different cellular pathways were affected by elevated temperature exposure in each compartment; Symbiodinium tended to up-regulate the expression of proteins involved in the cellular stress response, whereas the differentially expressed host coral proteome featured numerous proteins involved in cytoskeletal structure, immunity, and metabolism. These proteome-scale data suggest that the coral host and its intracellular dinoflagellates have differing cellular strategies for acclimating to elevated temperatures.

The operating parameters that affect the performance of the online preconcentration technique "analyte focusing by micelle collapse-MEKC (AFMC-MEKC)" were examined using a multivariate approach involving experimental design to determine the sunscreen agents in cosmetics. Compared to the single-variable approach, the advantage of the multivariate approach was that many factors could be investigated simultaneously to obtain the best separation condition. A fractional factorial design was used to identify the fewest significant factors in the central composite design (cCD). The cCD was adopted for evaluating the location of the minimum or maximum response in this study. The influences of the experimental variables on the response were investigated by applying a chromatographic exponential function. The optimized condition and the relationship between the experimental variables were acquired using the JMP software. The ANOVA analysis indicated that the Tris pH value, SDS concentration, and ethanol percentage influenced the separation quality and significantly contributed to the model. The optimized condition of the running buffer was 10 mM Tris buffer (pH 9.5) containing 60 mM SDS, 7 mM γ-CD, and 20% v/v ethanol. The sample was prepared in 100 mM Tris buffer (pH 9.0) containing 7.5 mM SDS and 20% v/v ethanol. The SDS concentration in the sample matrix was slightly greater than the CMC value that makes the micelle be easily collapsed and the analytes be accumulated in the capillary. In addition, sunscreen agents in cosmetics after 1000-fold dilution were successfully determined by AFMC-MEKC.

Serum vitamin D is bound to vitamin D-binding protein (DBP). We studied the roles of DBP in streptozotocin-diabetic rats and high glucose (HG)-cultured cells. In diabetic rat sera, there was one upregulated (with a lower isoelectric point [pI], phosphorylated at S268, S270, S464 and T269) and one downregulated (with a higher pI, phosphorylated at S454 and S457) DBP. DBP levels with lower pI were increased in diabetic rat kidney and liver. HG (30 mM) increased DBP protein expression in NRK-49F cells and Clone-9 hepatocytes. HG decreased pI of DBP in Clone-9 hepatocytes. Moreover, DBP short hairpin ribonucleic acid attenuated 1,25-(OH)2D3-induced attenuation of HG-induced renin (but not collagen IV and fibronectin) protein expression in NRK-49F cells. Thus, DBP level is increased whereas DBP is phosphorylated in diabetic rat serum. HG increased DBP protein expression in renal fibroblasts and hepatocytes. Moreover, DBP is required for vitamin D-induced attenuation of HG-induced renin in NRK-49F cells.

While a base substitution in intron 4 of GLA (IVS4+919G>A) that causes aberrant alternative splicing resulting in Fabry disease has been reported, its molecular mechanism remains unclear. Here we reported that upon IVS4+919G>A transversion, H3K36me3 was enriched across the alternatively spliced region. PSIP1, an adapter of H3K36me3, together with Hsp70 and NONO were recruited and formed a complex with SF2/ASF and SRp20, which further promoted GLA splicing. Amiloride, a splicing regulator in cancer cells, could reverse aberrant histone modification patterns and disrupt the association of splicing complex with GLA. It could also reverse aberrant GLA splicing in a PP1-dependant manner. Our findings revealed the alternative splicing mechanism of GLA (IVS4+919G>A), and a potential treatment for this specific genetic type of Fabry disease by amiloride in the future.

Valproic acid (VA) is a branch chain fatty acid that is widely used to treat epilepsy and convulsion. Recent studies show that VA can also be used to treat migraine headaches, bipolar disorder, and other diseases such as Alzheimer disease. However, clinical treatment with VA may cause hepatotoxicity, bone marrow suppression, and hyperammonemic encephalopathy. Valproic acid is also a known human teratogen. Because of the potential cytotoxic effects of VA and its major metabolite, 2-propyl 4-pentenoic acid (4-ene VA), VA plasma concentrations must be closely monitored during clinical applications of VA in order to avoid severe side effects. This study developed a derivatization oriented strategy for increasing sensitivity in detecting VA in quantities as low as 20μL and its metabolites in human plasma. After micro-scale liquid-liquid extraction (MLLE) and micro-scale derivatization, VA and 4-ene VA were quantitated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The linear ranges were 10-1000μM for VA and 5-500μM for 4-ene VA. All relative standard deviation (RSD) and relative error (RE) values obtained in intra- and inter-day analyses of VA and 4-ene VA were below 8%. The structures of VA and its metabolite derivatives were further identified by nano ultra performance liquid chromatographic system (nanoUPLC) coupled with tandem mass spectrometry (MS/MS). Since protein modifications induced by VA were also identifiable by nanoUPLC-MS/MS, these modifications may be useful biological indicators of a toxic reaction during clinical applications of VA.

Background Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed. Methods Circulating CD14+CD16+ of breast cancer patients and induced CD14+CD16+ DCs were identified as tDCs by treating CD14+ monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo. Results The CD14+CD16+ tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c+ DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c+/Ly6G+/IL-10+ in the BALB/c mice. Conclusions Galectin-1 can induce tDCs after internalizing into CD14+ monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy. General significance Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development.

Spermidine and its derivative, spermine, are basic compounds with unique roles in physiological function. This study used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to monitor these analytes in human urine and blood. The MALDI-TOF MS is also suitable when a high-throughput analytical method is required. Unlike liquid chromatography (LC)-MS, MALDI-TOF MS does not require a mobile phase in sample separation and generates very little organic waste. Micro liquid-liquid extraction was also performed to minimize the use of organic solvents in this study. After alkalization step, biosamples were prepared by adding a small volume (20μL) of organic solvent to concentrate, and purify the spermidine and spermine contained in the urine and blood samples. A suitable extraction protocol was developed after optimizing various conditions associated with extraction efficiency. The proposed method was then successfully used to monitor these compounds in human urine (mean value1μg/mL) and blood (mean value1μg/mL).

A simple and sensitive capillary zone electrophoresis (CZE) with UV detection (214 nm) was developed and validated for the simultaneous determination of the acetylcholinesterase inhibitors (AChEI), donepezil, and rivastigmine, with antipsychotic drugs in plasma. A sample pretreatment by liquid–liquid extraction and subsequent quantification by CZE with field-amplified sample injection (FASI) was used. The optimum separation for these analytes was achieved in

Transforming growth factor-β (TGF-β) is pivotal in diabetic nephropathy (DN). Angiotensin converting enzyme-2 (ACE2) converts angiotensin II (Ang II) to angiotensin 1-7 (Ang-(1-7)), which binds to Mas. Proximal tubular ACE2 is decreased in DN. ACE2 deficiency exacerbates whereas ACE2 overexpression attenuates DN. Thus, we investigated the mechanism of high glucose-decreased ACE2 in terms of the interaction between TGF-β and ACE2-Ang-(1-7)-Mas in NRK-52E cells. We found that high glucose increased TGF-β1. SB431542 attenuated high glucose-inhibited ACE2 and Mas and Ang-(1-7) conversion from Ang II while attenuating high glucose-induced fibronectin. TGF-β1 also decreased ACE2 and Mas and Ang-(1-7) conversion from Ang II. A779 attenuated Ang-(1-7)-decreased TGF-β1 and Ang-(1-7)-activated JAK2-STAT3. Moreover, A779, LY294002 and AG490 attenuated Ang-(1-7)-inhibited TGF-β1. The combination of Ang-(1-7) and Mas attenuated TGF-β1 (but not high glucose)-induced fibronectin. Thus, high glucose decreases ACE2 via TGF-βR in NRK-52E cells. Additionally, there is a negative feedback function between TGF-β and ACE2, and the combined inhibition of TGF-β and activation of the ACE2-Ang-(1-7)-Mas may be useful for treating diabetic renal fibrosis.

This study examines the use of unmodified magnetite nanoparticles (Fe(3)O(4) NPs) for selective extraction and enrichment of the catecholamines dopamine (DA), noradrenaline (NE), and adrenaline (E), prior to analysis using capillary electrophoresis with UV detection. Coordination between Fe(3+) on-the-surface Fe(3)O(4) NPs and the catechol moiety of catecholamines enables Fe(3)O(4) NPs to capture catecholamines from an aqueous solution. We obtained maximum loading of catecholamines on the NP surface by adjusting the pH of the solution to 7.0. In addition, catecholamine loading on the Fe(3)O(4) NPs increased in conjunction with NP concentrations. H(3)PO(4) was found to be efficient for the removal of adsorbed catecholamines on the NP surface. Adding 1.2% poly(diallyldimethylammonium chloride) to the background electrolyte resulted in a baseline separation of the liberated catecholamines within 20 min. Under optimal extraction and separation conditions, the limit of detections at a S/N ratio of 3 for E, NE, and DA were 9, 8, and 10 nM, respectively. Significantly, the combination of a phenylboronate-containing spin column and the proposed method was successfully applied to the determination of NE and DA in human urine and NE in Portulaca oleracea L. leaves.

Gastrodermal lipid bodies (LBs) are organelles involved in the regulation of the mutualistic I endosymbiosis between reef-building corals and their dinoflagellate endosymbionts (genus Symbiodinium). As their molecular composition remains poorly defined, we herein describe the first gastrodermal LB proteome and examine in situ morphology of LBs in order to provide insight into their structure and function. After tissue separation of the tentacles of the stony coral Euphyllia glabrescens, buoyant LBs of the gastroderm encompassing a variety of sizes (0.5―4 μm in diameter) were isolated after two cycles of subcellular fractionation via stepwise sucrose gradient ultracentrifugation and detergent washing. The purity of the isolated LBs was demonstrated by their high degree of lipid enrichment and as well as the absence of contaminating proteins of the host cell and Symbiodinium. LB-associated proteins were then purified, subjected to SDS-PAGE, and identified by MS using an LC-nano-ESI-MS/ MS. A total of 42 proteins were identified within eight functional groups, including metabolism, intracellular trafficking, the stress response/molecular modification and development. Ultrastructural analyses of LBs in situ showed that they exhibit defined morphological characteristics, including a high-electron density resulting from a distinct lipid composition from that of the lipid droplets of mammalian cells. Coral LBs were also characterized by the presence of numerous electron-transparent inclusions of unknown origin and composition. Both proteomic and ultrastructural observations seem to suggest that both Symbiodinium and host organelles, such as the ER, are involved in LB biogenesis.

Gemcitabine (2'-deoxy-2', 2'-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27(Kip1) protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27(Kip1) protein degradation. The induction of UBE2M and reduction of p27(Kip1) by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27(Kip1). Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27(Kip1). Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27(Kip1) expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27(Kip1) in drug resistance.

The renin-angiotensin system (RAS) participates in regulating the body fluid, such as angiotensin II (1-8), and plays an important role in blood pressure control. In RAS, angiotensin II (1-8) is the major effector but other angiotensins may also mediate the actions of the RAS. In this study, a simple method is proposed for analysis of the metabolites of angiotensin II (1-8). Experimental data was acquired by a nanoLC system connected with quadruple time-of-flight (Q-TOF) mass spectrometry. The flow rate of this method was at 400 nL/min, and this nanoliter level flow rate would lower the consumption of organic solvents.

A simple and micro-scale liquid chromatographic (LC) method coupled with mass spectrometry was developed for analyzing major proteins in human urine. After one-step sample preparation, proteins were precipitated, redissolved, and digested. Only micro-liter level (10 μL) of urine was sufficient for major protein identification. This method was applied in clinical study, and urine proteins were monitored after medication with angiotensin-converting enzyme inhibitor within 24 hr. This method can identify many important proteins in human urine. Bioactive peptides associated with blood pressure control can also be identified simultaneously. We hope this simple method may prove useful in clinical research and disease diagnosis.

Although accelerated atherosclerosis and arteriosclerosis are the main causes of cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients, the molecular pathogenesis remains largely obscure. Our study of the aortic function in a typical CKD model of subtotal nephrectomy (SNX) rats demonstrated phenotypes that resemble CKD patients with aortic stiffness. The 2-DE analysis of rat aortas followed by MS identified 29 up-regulated and 53 down-regulated proteins in SNX rats. Further Western blot and immunohistochemistry analyses validated the decreased HSP27 and increased milk fat globule epidermal growth factor-8 (MFG-E8) in SNX rats. Functional classification of differential protein profiles using KOGnitor revealed that the two major categories involved in aortic stiffness are posttranslational modification, protein turnover, chaperones (23%) and cytoskeleton (21%). Ingenuity Pathway Analysis highlighted cellular assembly and organization, and cardiovascular system development and function as the two most relevant pathways. Among the identified proteins, the clinical significance of the secreted protein MFG-E8 was confirmed in 50 CKD patients, showing that increased serum MFG-E8 level is positively related to aortic stiffness and renal function impairment. Drug interventions with an inhibitor of the angiotensin converting enzyme, enalapril, in SNX rats improved aortic stiffness and decreased MFG-E8 depositions. Together, our studies provide a repertoire of potential biomarkers related to the aortic stiffness in CKD.

Symbiosomes are specific intracellular membrane-bound vacuoles containing microalgae in a mutualistic Cnidaria (host)-dinoflagellate (symbiont) association. The symbiosome membrane is originally derived from host plasma membranes during phagocytosis of the symbiont; however, its molecular components and functions are not clear. In order to investigate the protein components of the symbiosome membranes, homogenous symbiosomes were isolated from the sea anemone Aiptasia pulchella and their purities and membrane intactness examined by Western blot analysis for host contaminants and microscopic analysis using various fluorescent probes, respectively. Pure and intact symbiosomes were then subjected to biotinylation by a cell impermeant agent (Biotin-XX sulfosuccinimidyl ester) to label membrane surface proteins. The biotinylated proteins, both Triton X-100 soluble and insoluble fractions, were subjected to 2-D SDS-PAGE and identified by MS using an LC-nano-ESI-MS/MS. A total of 17 proteins were identified. Based on their different subcellular origins and functional categories, it indicates that symbiosome membranes serve as the interface for interaction between host and symbiont by fulfilling several crucial cellular functions such as those of membrane receptors/cell recognition, cytoskeletal remodeling, ATP synthesis/proton homeostasis, transporters, stress responses/chaperones, and anti-apoptosis. The results of proteomic analysis not only indicate the molecular identity of the symbiosome membrane, but also provide insight into the possible role of symbiosome membranes during the endosymbiotic association.

Arecoline is the main alkaloid present in the areca nut (or betel nut) and it has central nervous system effects. Its pharmacological activities induce the constriction of the bronchial smooth muscles, and stimulation of the lacrimal and intestinal glands. Chewing areca nut is harmful to health because this habit may increase the risk of the development of oral cancer. In this study, a fast method was provided for the determination of areca alkaloids by matrix-assisted laser desorption ionization (MALDI) mass spectrometer with a time-of-flight (TOF) analyzer. Traditionally the MALDI-TOF method was not suitable for the analysis of small molecular weight (m/z600) compounds because of the high background of the matrix. In this study, a new matrix was utilized to decrease the background interference effectively. After simple sample preparation, 1 microL sample supernatant was mixed with 1 microL matrix and then deposited on the target plate. This new matrix was also used to test the MALDI imaging experiment. Application of this MALDI-TOF method for trace analysis of arecoline by this new matrix in human plasma at sub microM level proved workable.

(G:3-7)-dendri-PAMAM-(APO-Phe-Lys)(x) (2, APO = aminopropanol, Phe = phenylalanine, Lys = lysine) were prepared and used in a binding study with pyridoxal 5'-phosphate. The results revealed a positive dendritic effect, and binding ability was found to vary with the environment. (G:3-7)-dendri-PAMAM-(APO-Phe-Lys)(x) (2) demonstrated better binding ability at higher pH, and protonation of lysine was considered to affect binding. The strongest binding affinity was K(b) = 254.3 mM(-1) at pH 9, which was shown by (G:7)-dendri-PAMAM-(APO-Phe-Lys)(490) (2e).

A simple nanoscale liquid chromatographic method with tandem mass spectrometry (LC‐MS‐MS) was developed for the determination of very basic compounds, biguanides, such as metformin and phenformin. Plasma samples were deproteinated and acidified with heptafluorobutyric acid (HFBA) and the resulting supernatant injected into the nanoscale separation system. Chromatography was carried out using a short reversed‐phase C18 column under isocratic elution with methanol‐40 mM aqueous HFBA (75∶25, v/v) at a flow rate of 800 nL/min. Therapeutic monitoring on metformin and phenformin to minimize adverse side effects is important. The main advantage of the nanoscale method is the lower consumption of organic solvent. This nanoscale method is suitable for the investigation and monitoring of the concentration of oral antihyperglycemic drugs in clinical patients.

In typical mass spectrometry-based protein identification using peptide fragmentation fingerprinting, front-end separation plays a critical role in successful peptide sequencing. This separation step demands a great deal of time and usually is the rate-limiting step for the whole process. Here we provide an alternative separation method, based on a simple nanoflow delivery system, that is able to shorten the separation time considerably. This system consists of a 25-μl syringe connected to a manually packed reversed-phase mini-capillary column that can be directly coupled to an electrospray ionization tandem mass spectrometer. A syringe pump is then used to deliver the peptide mixtures at a nanoscale flow rate. We examined the efficiency and efficacy of this method by analyzing the tryptic peptides of bovine serum albumin and of 10 Escherichia coli proteins separated by two-dimensional gel electrophoresis (2DE). The results showed that identification of each protein could be achieved successfully within 25 min by using the disposable mini-capillary column. Moreover, all 2DE-separated E. coli proteins were identified at high confidence levels. Together, our data suggest that this method is a suitable option for mass spectrometry-based protein identification.

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.

The metabolites of environmental chemicals play key roles in carcinogenesis. Areca nut is strongly associated with the development of oral potentially malignant disorders (OPMD) or cancer. The main alkaloid in the areca nut is arecoline, which is highly cytotoxic and genotoxic. Arecoline N-oxide, a metabolite of areca nut alkaloids, which has been identified in animal urine, has been shown to induce mutagenicity in bacteria. In this study, it was found that its protein adduct could be detected in oral keratinocytes treated with areca nut extract. Increased collagen expression and severity of squamous hyperplasia were observed in arecoline N-oxide treated mice. In cultured oral fibroblasts, arecoline N-oxide showed stronger effects on the increase of fibrotic related genes including TGF-beta1, S100A4, MMP-9, IL-6, and fibronectin and a decrease of E-cadherin as compared with arecoline. Finally, arecoline N-oxide stimulation effectively increased the DNA damage marker, gamma-H2A.X, both in vitro and in vivo. Taken together, these results indicate that arecoline N-oxide shows a high potential for the induction of OPMD.

In continuing the search for fluorescent reagents for analytical derivatization in chromatography, we found a simple chemical, (2-naphthoxy)acetyl chloride, with potential fluorophore/chromophore characteristics for the highly sensitive detection of analytes with an amino function. The reagent has an auxochrome (a substituted alkoxy moiety) attached to the fluorophoric/chromophoric naphthalene system, resulting in favorable spectrophotometric properties. The reagent can be easily prepared from (2-naphthoxy)acetic acid and has been used in organic synthesis; it is initially introduced as a fluorescent reagent to derivatise amantadine and memantine (amino pharmaceuticals) as model analytes. The resulting naphthoxy derivatives of the drugs can be analyzed at sub-microM levels by HPLC with fluorimetric detection (excitation wavelength 227 nm, emission wavelength 348 nm). Application of the reagent to the fluorimetric derivatization of important biological amines for sensitive detection can be expected.

Spermine and spermidine are multiple-nitrogen compounds found in many foods. Both compounds are essential for cell growth and human health. This study established a simple and fast method of detecting spermine and spermidine in food samples by matrix-assisted laser desorption/ionization combined with time-of-flight mass spectrometry (MALDI-TOF MS). After a simple sample preparation procedure, spermine and spermidine were directly detected by MALDI-TOF MS with no additional purification procedure. The calibration curves for spermine and spermidine ranged from 0.1 to 10 μg/mL. In intra- and inter-batch assays of three different concentrations of spermine and spermidine, all relative standard deviations and relative errors were below 18.9%. These experimental results confirmed the practicability and effectiveness of the proposed MALDI-TOF MS method for fast determination of spermine and spermidine in food samples. Furthermore, since spermine and spermidine have important roles in apoptosis, up-regulation and down-regulation of spermine and spermidine during apoptosis were analyzed. After treating NRK-52E cells with spermine and spermidine, the cells were lysed, and cell proteins were collected, and digested. Apoptosis-related proteins were then identified by tandem MS.

A simple and sensitive method is described for the determination of biologically important fatty acids including dodecanoic, tetradecanoic, hexadecanoic, octadecanoic, hexadecenoic, octadecenoic and octadecadienoic acids. The method is based derivatization with (2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES) in toluene using potassium carbonate and 18-crown-6 as reaction activators. The resulting naphthoxyethyl derivatives were simply analyzed by isocratic HPLC with fluorometric detection (λex=235 nm, λem=350 nm). The excess fluorescence reagent (NOEPES) was readily removed after derivatization by a quick acid treatment. This minimizes the interference of excess NOEPES in fluorometric detection. The lower limit of quantitation 50 nM with a detection limit (S/N=3) of about 7.5 nM (or 0.15 pmol per 20 μL injection). Application of the method to analysis of free fatty acids in plasma proved feasible (using only 10 μL of plasma sample).

A simple and sensitive liquid chromatographic method is described for the simultaneous determination of biologically important very long chain fatty acids (docosanoic, tetracosanoic and hexacosanoic acids) as fluorogenic derivatives. The method is based on the derivatization of the fatty acids with 2-(2-naphtoxy)ethyl 2-(piperidino)ethanesulfonate (NOEPES) in toluene in the presence of potassium carbonate and 18-crown-6. Several parameters affecting the derivatization were studied, including reaction temperature, reaction time, reaction solvent, base catalyst and the amount of the reagent. The resulting derivatives were analyzed by HPLC with fluorimetric detection (λex=235 nm; λem=366 nm). The linear range for the determination of docosanoic, tetracosanoic and hexacosanoic acids was 0.028–1.4 μM with a detection limit of about 5.6 nM (S/N=3) (56 fmol per 10 μL injection). Application of the method to the analysis the non-esterified (free) very long chain fatty acids spiked in plasma proved feasible.

Chitosan nanoparticle, a biocompatible material, was used as a potential drug delivery system widely. Our current investigation studies were the bioeffects of the chitosan nanoparticle uptake by liver cells. In this experiment, the characterizations of chitosan nanoparticles were measured by transmission electron microscopy and particle size analyzer. The average size of the chitosan nanoparticle was224.6±11.2 nm, and the average zeta potential was+14.08±0.7 mV. Moreover, using proteomic approaches to analyze the differential protein expression patterns resulted from the chitosan nanoparticle uptaken by HepG2 and CCL-13 cells identified several proteins involved in the PI3K/AKT1/mTOR pathway. Our experimental results have demonstrated that the chitosan nanoparticle may involve in the liver cancer cell metastasis and proliferation.

Protein phosphorylation is an important regulatory post-translational modification in many biochemical processes. The phosphopeptide analysis strategies developed in this study were all at microscale. After using a standard microwave oven to assist protein digestion, phosphoserine and phosphothreonine were tagged with chemical analogues, such as 2-mercaptoethanol and 3-mercapto-1-propanol, to enable simultaneously relative quantitation and identification. This method enabled the use of thio alcohols for direct labeling of phosphorylated sites (not labeled at the mercapto, amino, hydroxyl, or carboxyl groups) of phosphopeptides. Various digestion parameters (e.g., microwave power, reaction time, NH4HCO3 concentration) and derivatization efficiency parameters (e.g., reaction time, labeling tag concentration) were studied and optimized. In both control and experimental samples, microwave-assisted digestion coupled with relative quantitation using analogue tags enabled calculation of phosphopeptide ratios in the same sequence. A non-labeling method was also established for quantifying phosphopeptides in human plasma by using the abundant protein albumin as an internal control for normalizing relative quantities of phosphopeptides. Nano ultra-performance liquid chromatography (nanoUPLC) was combined with LTQ Orbitrap to enable simultaneous protein relative quantitation and identification. These strategies proved to be effective for quantifying phosphopeptides in biological samples.

Oil bodies formed in Auxenochlorella protothecoides induced during limited nutrition had a coating of caleosin. The total lipid content obtained from A. protothecoides in unstressed cultures (first week) was ~210 mg/g compared to the 231 mg/g obtained in the third week (nutrient limited) and 290 mg/g obtained in the fourth week (nutrient limited). The proportion of total saturated fatty acids increased from 28 to 46 %, whereas that of total polyunsaturated fatty acids decreased from 52 to 35 %. The expression levels of the 28 kDa caleosin protein in A. protothecoides rose to a maximum up to 4 weeks; immunolocalization studies showed that caleosin was predominantly associated with the membranes of oil bodies.

Background Samples originating from body fluids often contain a complex mixture of inorganic salts, buffers, chaotropic agents, surfactant/detergents, preservatives, and other solubilizing agents. The presence of those contaminants often precludes direct analysis by mass spectrometry. Urine, a blood filtrate produced by the urinary system, is an ideal bio-sample and a rich source of biomarkers for diagnostic information. Methods To enhance our understanding of urinary proteome, the urine proteins were prepared by magnetic nanoparticles (MNPs) combined with MACS separation column system and then identified by reverse phase nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (RP-nano-HPLC-ESI-MS/MS) followed by peptide fragmentation pattern. Results Experimental results have revealed that the better protein identification for the demonstration of bovine serum albumin (BSA) in artificial urine. Using this cleanup approach, a total of 542 peptides, corresponding to 282 unique proteins, were identified from human urine samples, in which 54 proteins have higher confidence levels. Indeed, this study has revealed that some biological factors might be increased along with aging, such as up-regulation of immunoproteins. Conclusions The present study was designed to establish optimal techniques to develop a proteomic map of urinary proteins, and a cleanup method that greatly simplifies this sample preparation process was proposed.

The cell responses to biopolymer surface at the early adhesion stages can be critical for cell survival. The purpose of this research was to assess formation of hyaluronic acid (HA) biopolymer surface, the fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer materials using a mass spectrometry-based profiling system. Surfaces were covered by multi-walled carbon nanotubes (CNT), chitosan (CS), and HA to increase the surface area, enhance the adhesion of biopolymer and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNT/CS/HA electrodes of quartz crystal microbalance (QCM) were greatly exceeded those on other surfaces that were consistent with cell-count technique. Moreover, analyzing differential protein expressions of adhered fibroblasts on those biopolymer surfaces by proteomic approaches identified CD36, CD44, PP2A, and CDK9 as key proteins. To validate the influences of those four proteins on adhesions of fibroblasts on biopolymers, the cells were blocked by antibodies of the proteins and the adhesions of cells on the tested biopolymer surfaces were examined using a QCM technique, flow cytometric analysis and morphological observations. The results of significantly decreasing the weights and densities of the blocked fibroblasts adhering to CNT/CS/HA surfaces were obtained, and validate those proteins found by proteomic approaches. Utilizing mass spectrometry-based proteomics to evaluate cell adhesions on biopolymers is proposed.

The renin–angiotensin–aldosterone system (RAAS) is an essential body fluid maintenance system that controls pressure in the human body. The conversion of angiotensin I to angiotensin II by angiotensin-converting enzyme (ACE) is a key process in the RAAS because angiotensin II causes the vasoconstriction association with hypertension. Because of its effectiveness as an ACE blocker, quinipril is widely used for clinical treatment of hypertension and chronic congestive heart failure . Matrix-assisted laser desorption/ionization coupled with time-of-flight analyzer (MALDI-TOF) is a high throughput instrument for biological sample analysis. This study developed a micro-scale approach for using MALDI-TOF to detect quinapril in biological samples. A micro-liquid-liquid-extraction strategy combined with ion-pair interaction successfully extracted quinapril from aqueous layer to organic layer. Quinolones were then used as matrix additives to suppress undesired substances in plasma produce signals. Several factors affecting extraction efficiency were investigated in a biosample with a volume of only 10 μL. This method is successful to monitor quinapril in the clinical therapeutic range. The proposed method proved effective for monitoring the trace amounts of quinapril typically used for clinical therapy. The relative standard deviation (R.S.D.) and relative error (R.E.) used for evaluating within- and between-day assays of quinapril in plasma consistently remained below 15%.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been broadly applied to analyze high-molecular-weight compound (such as polymer or proteomic research) but seldom used for low-molecular-weight compound analysis. The objective of this study is the development of a simple analytical method for the determination of the concentration of tripeptide glutathione (GSH) by MALDI-TOF MS. Unfortunately, GSH could not be detected directly by MALDI-TOF MS. Our method is based on the derivatization of GSH with 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) in acetonitrile using potassium hydroxide (KOH) as a base catalyst. After simple extraction step, the supernatant is spotted on a target plate, mixed with matrix α-cyano-4-hydroxycinnamic acid (CHCA) and then detected by MALDI-TOF MS. Some parameters affecting the derivatization of GSH were investigated, such as the concentration of BrDMC, KOH, different base catalyst, and reaction time, etc. The regression equations of GSH derivative possessed good linearity (r≧0.995) over the range of 1.0-100.0 μM. The relative standard deviation (R.S.D.) and relative error (R.E.) values in intra- and inter-day assays were below 13%, which showed good precision and accuracy. This proposed method was successfully applied to monitor the concentration of GSH in human blood at micro-scale level.

Environmental and food-borne acrylamide is a suspected carcinogen in humans and is associated with several cancer types. Its biological metabolite, glycidamide, is also harmful to human health. The presence of acrylamide in the living environment makes this toxic chemical an important public health issue. Acrylamide and glycidamide bind with proteins to form protein adducts in metabolic processes. These metabolic adducts can be considered environmental modifications of proteins. This study used a simple proteomic strategy to identify acrylamide and glycidamide adducts bound in major plasma proteins. After simple sample preparation, new protein modifications by acrylamide and glycidamide were identified using nano LC combined with quadruple time-of-flight (Q-TOF) mass spectrometry. This method required only 10 μL of human plasma sample for protein modification survey. Hopefully, this strategy can help to discover protein–acrylamide (or glycidamide) adducts that are biomarkers of human exposure to high-dose acrylamide. These biomarkers may also elucidate the metabolic pathways of acrylamide and glycidamide.

We establish a triple-stacking capillary electrophoresis (CE) separation method to monitor methotrexate (MTX) and its eight metabolites in cerebrospinal fluid (CSF). Three stacking methods with different mechanisms were combined and incorporated into CE separation. Complete stacking and sharp peaks were achieved. Firstly, the optimized buffer (60 mM phosphate containing 15% THF and 100 mM SDS) was filled into the capillary, which was followed by the higher conductivity buffer (100 mM phosphate, 2 psi for 45 s). The analytes extracted from CSF were injected at 2 psi for 99.9 s, which provided long sample zones and pH junction for focusing. Finally, the stacking step was performed by sweeping, and separation was achieved by micellar electrokinetic chromatography. The results of the linear regression equations indicated high linearity (r ≥ 0.9981) over the range of 0.5–7 μM. In intra- and inter-batch results, all data of RSD and RE were below 11%, indicating good precision and accuracy of this method. The LODs (S/N = 3) were 0.1 μM for MTX, 7-hydroxymethotrexate (7-OHMTX) and MTX-polyglutamates (MTX-(Glu)n, n = 2–5), 0.2 μM for MTX-(Glu)6, and 0.3 μM for 2,4-diamino-N 10-methylpteroic acid (DAMPA) and MTX-(Glu)7. Our method was implemented for analysis of MTX and its metabolites in the CSF, and could be used for evaluation of its curative effects of acute lymphoblastic leukemia patients. The data were also confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results showed good coincidence.