Samanthi Wickramasekara

Isolation and identification of antioxidant peptides from enzymatically hydrolyzed rice bran protein.

Khao Dawk Mali 105 rice bran protein (RBP) was fractionated into albumin (12.5%), globulin (13.9%), glutelin (70.8%) and prolamine (2.9%). The native and denatured RBP fractions were hydrolyzed with papain and trypsin for 3h at optimum conditions. The RBP fractions and their hydrolysates were evaluated for their antioxidant activity by the Oxygen Radical Absorbance Capacity (ORAC) assay. The trypsin-hydrolyzed denatured albumin exhibited the highest antioxidant activity with an ORAC value of 4.07 μmol of Trolox equivalent (TE)/mg protein. This hydrolysate was separated by using RP-HPLC and three fractions with high antioxidant activity were examined by LTQ-FTICR ESI mass spectrometry. The MW of the peptides from these fractions were 800-2100 Da. and consisted of 6-21 amino acid residues. Most of the peptides from the fractions demonstrated typical characteristics of well-known antioxidant peptides. The results suggest that trypsin-hydrolyzed denatured rice bran albumin might be useful as a natural food antioxidant.

Electrospray Quadrupole Travelling Wave Ion Mobility Time-of-Flight Mass Spectrometry for the Detection of Plasma Metabolome Changes Caused by Xanthohumol in Obese Zucker (fa/fa) Rats

This study reports on the use of traveling wave ion mobility quadrupole time-of-flight (ToF) mass spectrometry for plasma metabolomics. Plasma metabolite profiles of obese Zucker fa/fa rats were obtained after the administration of different oral doses of Xanthohumol; a hop-derived dietary supplement. Liquid chromatography coupled data independent tandem mass spectrometry (LC-MSE) and LC-ion mobility spectrometry (IMS)-MSE acquisitions were conducted in both positive and negative modes using a Synapt G2 High Definition Mass Spectrometry (HDMS) instrument. This method provides identification of metabolite classes in rat plasma using parallel alternating low energy and high energy collision spectral acquisition modes. Data sets were analyzed using pattern recognition methods. Statistically significant (p < 0.05 and fold change (FC) threshold > 1.5) features were selected to identify the up-/down-regulated metabolite classes. Ion mobility data visualized using drift scope software provided a graphical read-out of differences in metabolite classes.

A multiplex biomarker approach for the diagnosis of transitional cell carcinoma from canine urine

Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a noninvasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts

BackgroundOsteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells.ResultsShotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines.ConclusionsThe results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

Ion mobility-enhanced MSE-based label-free analysis reveals effects of low-dose radiation post contextual fear conditioning training on the mouse hippocampal proteome

UNLABELLED Recent advances in the field of biodosimetry have shown that the response of biological systems to ionizing radiation is complex and depends on the type and dose of radiation, the tissue(s) exposed, and the time lapsed after exposure. The biological effects of low dose radiation on learning and memory are not well understood. An ion mobility-enhanced data-independent acquisition (MS(E)) approach in conjunction with the ISOQuant software tool was utilized for label-free quantification of hippocampal proteins with the goal of determining protein alteration associated with low-dose whole body ionizing radiation (X-rays, 1Gy) of 5.5-month-old male C57BL/6J mice post contextual fear conditioning training. Global proteome analysis revealed deregulation of 73 proteins (out of 399 proteins). Deregulated proteins indicated adverse effects of irradiation on myelination and perturbation of energy metabolism pathways involving a shift from the TCA cycle to glutamate oxidation. Our findings also indicate that proteins associated with synaptic activity, including vesicle recycling and neurotransmission, were altered in the irradiated mice. The elevated LTP and decreased LTD suggest improved synaptic transmission and enhanced efficiency of neurotransmitter release which would be consistent with the observed comparable contextual fear memory performance of the mice following post-training whole body or sham-irradiation. SIGNIFICANCE This study is significant because the biological consequences of low dose radiation on learning and memory are complex and not yet well understood. We conducted a IMS-enhanced MS(E)-based label-free quantitative proteomic analysis of hippocampal tissue with the goal of determining protein alteration associated with low-dose whole body ionizing radiation (X-ray, 1Gy) of 5.5-month-old male C57BL/6J mice post contextual fear conditioning training. The IMS-enhanced MS(E) approach in conjunction with ISOQuant software was robust and accurate with low median CV values of 0.99% for the technical replicates of samples from both the sham and irradiated group. The biological variance was as low as 1.61% for the sham group and 1.31% for the irradiated group. The applied data generation and processing workflow allowed the quantitative evaluation of 399 proteins. The current proteomic analysis indicates that myelination is sensitive to low dose radiation. The observed protein level changes imply modulation of energy metabolism pathways in the radiation exposed group, specifically changes in protein abundance levels suggest a shift from TCA cycle to glutamate oxidation to satisfy energy demands. Most significantly, our study reveals deregulation of proteins involved in processes that govern synaptic activity including enhanced synaptic vesicle cycling, and altered long-term potentiation (LTP) and depression (LTD). An elevated LTP and decreased LTD suggest improved synaptic transmission and enhanced efficiency of neurotransmitter release which is consistent with the observed comparable contextual fear memory performance of the mice following post-training whole body or sham-irradiation. Overall, our results underscore the importance of low dose radiation experiments for illuminating the sensitivity of biochemical pathways to radiation, and the modulation of potential repair and compensatory response mechanisms. This kind of studies and associated findings may ultimately lead to the design of strategies for ameliorating hippocampal and CNS injury following radiation exposure as part of medical therapies or as a consequence of occupational hazards.

HDAC6 activity is not required for basal autophagic flux in metastatic prostate cancer cells

Histone deacetylase 6 is a multifunctional lysine deacetylase that is recently emerging as a central facilitator of response to stress and may play an important role in cancer cell proliferation. The histone deacetylase 6-inhibitor tubacin has been shown to slow the growth of metastatic prostate cancer cells and sensitize cancer cells to chemotherapeutic agents. However, the proteins histone deacetylase 6 interacts with, and thus its role in cancer cells, remains poorly characterized. Histone deacetylase 6 deacetylase activity has recently been shown to be required for efficient basal autophagic flux. Autophagy is often dysregulated in cancer cells and may confer stress resistance and allow for cell maintenance and a high proliferation rate. Tubacin may therefore slow cancer cell proliferation by decreasing autophagic flux. We characterized the histone deacetylase 6-interacting proteins in LNCaP metastatic prostate cancer cells and found that histone deacetylase 6 interacts with proteins involved in several cellular processes, including autophagy. Based on our interaction screen, we assessed the impact of the histone deacetylase 6-inhibitor tubacin on autophagic flux in two metastatic prostate cancer cell lines and found that tubacin does not influence autophagic flux. Histone deacetylase 6 therefore influences cell proliferation through an autophagy-independent mechanism.

Analysis of autophagic flux in response to sulforaphane in metastatic prostate cancer cells

SCOPE The phytochemical sulforaphane (SF) has been shown to decrease prostate cancer metastases in a genetic mouse model of prostate carcinogenesis, though the mechanism of action is not fully known. SF has been reported to stimulate autophagy, and modulation of autophagy has been proposed to influence SF cytotoxicity; however, no conclusions about autophagy can be drawn without assessing autophagic flux, which has not been characterized in prostate cancer cells following SF treatment. METHODS AND RESULTS We conducted an investigation to assess the impact of SF on autophagic flux in two metastatic prostate cancer cell lines at a concentration shown to decrease metastasis in vivo. Autophagic flux was assessed by multiple autophagy related proteins and substrates. We found that SF can stimulate autophagic flux and cell death only at high concentrations, above what has been observed in vivo. CONCLUSION These results suggest that SF does not directly stimulate autophagy or cell death in metastatic prostate cancer cells under physiologically relevant conditions, but instead supports the involvement of in vivo factors as important effectors of SF-mediated prostate cancer suppression.

Electrospray Ionization Traveling Wave Ion Mobility Spectrometry Mass Spectrometry for the Analysis of Plant Phenolics: An Approach for Separation of Regioisomers

The use of ion-mobility spectrometry (IMS) coupled to mass spectrometry (IMS–MS) for biomolecule analyses has steadily increased over the past two decades, and is now applied to both proteomic and metabolomic investigations. This chapter describes the application of traveling-wave ion-mobility spectrometry–mass spectrometry (TWIMS–MS) to the analysis of a selection of bioactive phytochemicals used in dietary supplements. Applications include the analysis of grape seed proanthocyanidins and the structural characterization of bioactive constituents of dietary supplements using TWIMS-MS in conjunction with tandem mass spectrometry. We also discussed is the application of TWIMS-MS for the gas-phase mobility separation of structural isomers and the estimation of collision cross sections for a small selection of phenolic compounds from hop Recent applications of IMS–MS to a broad range of biomolecule measurements have demonstrated that IMS–MS has emerged as a powerful analytical technique capable of providing the separation space necessary to analyze highly complex samples. We give a perspective on emerging applications of IMS–MS for small molecule and biopolymer applications.The combination of devices that allow real-time monitoring of living systems using IMS–MS is an exciting avenue of facilitating system-biology experiments. The future of IMS–MS is bright and full of opportunities.