Publications by authors named "Mark R Emmett"

61 Publications

Associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following Si ion irradiation.

Sci Rep 2021 Jul 21;11(1):14899. Epub 2021 Jul 21.

Department of Behavioral Neuroscience, L470, Oregon Health & Science University, 3181SW Sam Jackson Park Road, Portland, OR, 97239, USA.

The space radiation environment consists of multiple species of charged particles, including Si ions, that may impact brain function during and following missions. To develop biomarkers of the space radiation response, BALB/c and C3H female and male mice and their F2 hybrid progeny were irradiated with Si ions (350 MeV/n, 0.2 Gy) and tested for behavioral and cognitive performance 1, 6, and 12 months following irradiation. The plasma of the mice was collected for analysis of miRNA levels. Select pertinent brain regions were dissected for lipidomic analyses and analyses of levels of select biomarkers shown to be sensitive to effects of space radiation in previous studies. There were associations between lipids in select brain regions, plasma miRNA, and cognitive measures and behavioral following Si ion irradiation. Different but overlapping sets of miRNAs in plasma were found to be associated with cognitive measures and behavioral in sham and irradiated mice at the three time points. The radiation condition revealed pathways involved in neurodegenerative conditions and cancers. Levels of the dendritic marker MAP2 in the cortex were higher in irradiated than sham-irradiated mice at middle age, which might be part of a compensatory response. Relationships were also revealed with CD68 in miRNAs in an anatomical distinct fashion, suggesting that distinct miRNAs modulate neuroinflammation in different brain regions. The associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following Si ion irradiation could be used for the development of biomarker of the space radiation response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-93869-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295277PMC
July 2021

The Great Deceiver: miR-2392's Hidden Role in Driving SARS-CoV-2 Infection.

bioRxiv 2021 Apr 27. Epub 2021 Apr 27.

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provides an exciting avenue towards antiviral therapeutics. From patient transcriptomic data, we have discovered a circulating miRNA, miR-2392, that is directly involved with SARS-CoV-2 machinery during host infection. Specifically, we found that miR-2392 was key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia as well as promoting many symptoms associated with COVID-19 infection. We demonstrate miR-2392 is present in the blood and urine of COVID-19 patients tested, but not detected in COVID-19 negative patients. These findings indicate the potential for developing a novel, minimally invasive, COVID-19 detection method. Lastly, using both human and hamster models, we have developed a novel miRNA-based antiviral therapeutic targeting miR-2392 that significantly reduces SARS-CoV-2 viability and may potentially inhibit a COVID-19 disease state in the host.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.04.23.441024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095194PMC
April 2021

Effects of Low Dose Space Radiation Exposures on the Splenic Metabolome.

Int J Mol Sci 2021 Mar 17;22(6). Epub 2021 Mar 17.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.

Future space missions will include a return to the Moon and long duration deep space roundtrip missions to Mars. Leaving the protection that Low Earth Orbit provides will unavoidably expose astronauts to higher cumulative doses of space radiation, in addition to other stressors, e.g., microgravity. Immune regulation is known to be impacted by both radiation and spaceflight and it remains to be seen whether prolonged effects that will be encountered in deep space can have an adverse impact on health. In this study, we investigated the effects in the overall metabolism of three different low dose radiation exposures (γ-rays, O, and Fe) in spleens from male C57BL/6 mice at 1, 2, and 4 months after exposure. Forty metabolites were identified with significant enrichment in purine metabolism, tricarboxylic acid cycle, fatty acids, acylcarnitines, and amino acids. Early perturbations were more prominent in the γ irradiated samples, while later responses shifted towards more prominent responses in groups with high energy particle irradiations. Regression analysis showed a positive correlation of the abundance of identified fatty acids with time and a negative association with γ-rays, while the degradation pathway of purines was positively associated with time. Taken together, there is a strong suggestion of mitochondrial implication and the possibility of long-term effects on DNA repair and nucleotide pools following radiation exposure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22063070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002539PMC
March 2021

Cs γ Ray and Si Irradiation Induced Murine Hepatocellular Carcinoma Lipid Changes in Liver Assessed by MALDI-MSI Combined with Spatial Shrunken Centroid Clustering Algorithm: A Pilot Study.

ACS Omega 2020 Oct 22;5(39):25164-25174. Epub 2020 Sep 22.

Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas 77555, United States.

Characterization of lipids by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is of great interest because not only are lipids important structural molecules in both the cell and internal organelle membranes, but they are also important signaling molecules. MALDI-MSI combined with spatial image segmentation has been previously used to identify tumor heterogeneities within tissues with distinct anatomical regions such as the brain. However, there has been no systematic study utilizing MALDI-MSI combined with spatial image segmentation to assess the tumor microenvironment in the liver. Here, we present that image segmentation can be used to evaluate the tumor microenvironment in the liver. In particular, to better understand the molecular mechanisms of irradiation-induced hepatic carcinogenesis, we used MALDI-MSI in the negative ion mode to identify lipid changes 12 months post exposure to low dose Si and Cs γ ray irradiation. We report here the changes in the lipid profiles of male C3H/HeNCrl mice liver tissues after exposure to irradiation and analyzed using the spatial shrunken centroid clustering algorithm. These findings provide valuable information as astronauts will be exposed to high-charge high-energy (HZE) particles and low-energy γ-ray irradiation during deep space travel. Even at low doses, exposure to these irradiations can lead to cancer. Previous studies infer that irradiation of mice with low-dose HZE particles induces oxidative damage and microenvironmental changes that are thought to play roles in the pathophysiology of hepatocellular carcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsomega.0c03047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542585PMC
October 2020

Comparative RNA-Seq transcriptome analyses reveal dynamic time-dependent effects of Fe, O, and Si irradiation on the induction of murine hepatocellular carcinoma.

BMC Genomics 2020 Jul 1;21(1):453. Epub 2020 Jul 1.

Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77550, USA.

Background: One of the health risks posed to astronauts during deep space flights is exposure to high charge, high-energy (HZE) ions (Z > 13), which can lead to the induction of hepatocellular carcinoma (HCC). However, little is known on the molecular mechanisms of HZE irradiation-induced HCC.

Results: We performed comparative RNA-Seq transcriptomic analyses to assess the carcinogenic effects of 600 MeV/n Fe (0.2 Gy), 1 GeV/n O (0.2 Gy), and 350 MeV/n Si (0.2 Gy) ions in a mouse model for irradiation-induced HCC. C3H/HeNCrl mice were subjected to total body irradiation to simulate space environment HZE-irradiation, and liver tissues were extracted at five different time points post-irradiation to investigate the time-dependent carcinogenic response at the transcriptomic level. Our data demonstrated a clear difference in the biological effects of these HZE ions, particularly immunological, such as Acute Phase Response Signaling, B Cell Receptor Signaling, IL-8 Signaling, and ROS Production in Macrophages. Also seen in this study were novel unannotated transcripts that were significantly affected by HZE. To investigate the biological functions of these novel transcripts, we used a machine learning technique known as self-organizing maps (SOMs) to characterize the transcriptome expression profiles of 60 samples (45 HZE-irradiated, 15 non-irradiated control) from liver tissues. A handful of localized modules in the maps emerged as groups of co-regulated and co-expressed transcripts. The functional context of these modules was discovered using overrepresentation analysis. We found that these spots typically contained enriched populations of transcripts related to specific immunological molecular processes (e.g., Acute Phase Response Signaling, B Cell Receptor Signaling, IL-3 Signaling), and RNA Transcription/Expression.

Conclusions: A large number of transcripts were found differentially expressed post-HZE irradiation. These results provide valuable information for uncovering the differences in molecular mechanisms underlying HZE specific induced HCC carcinogenesis. Additionally, a handful of novel differentially expressed unannotated transcripts were discovered for each HZE ion. Taken together, these findings may provide a better understanding of biological mechanisms underlying risks for HCC after HZE irradiation and may also have important implications for the discovery of potential countermeasures against and identification of biomarkers for HZE-induced HCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-020-06869-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329445PMC
July 2020

Efficient identification of multiple pathways: RNA-Seq analysis of livers from Fe ion irradiated mice.

BMC Bioinformatics 2020 Mar 20;21(1):118. Epub 2020 Mar 20.

Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas, USA.

Background: mRNA interaction with other mRNAs and other signaling molecules determine different biological pathways and functions. Gene co-expression network analysis methods have been widely used to identify correlation patterns between genes in various biological contexts (e.g., cancer, mouse genetics, yeast genetics). A challenge remains to identify an optimal partition of the networks where the individual modules (clusters) are neither too small to make any general inferences, nor too large to be biologically interpretable. Clustering thresholds for identification of modules are not systematically determined and depend on user-settable parameters requiring optimization. The absence of systematic threshold determination may result in suboptimal module identification and a large number of unassigned features.

Results: In this study, we propose a new pipeline to perform gene co-expression network analysis. The proposed pipeline employs WGCNA, a software widely used to perform different aspects of gene co-expression network analysis, and Modularity Maximization algorithm, to analyze novel RNA-Seq data to understand the effects of low-dose Fe ion irradiation on the formation of hepatocellular carcinoma in mice. The network results, along with experimental validation, show that using WGCNA combined with Modularity Maximization, provides a more biologically interpretable network in our dataset, than that obtainable using WGCNA alone. The proposed pipeline showed better performance than the existing clustering algorithm in WGCNA, and identified a module that was biologically validated by a mitochondrial complex I assay.

Conclusions: We present a pipeline that can reduce the problem of parameter selection that occurs with the existing algorithm in WGCNA, for applicable RNA-Seq datasets. This may assist in the future discovery of novel mRNA interactions, and elucidation of their potential downstream molecular effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12859-020-3446-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082965PMC
March 2020

Differentiation of 2-hydroxyglutarate enantiomers and its lactones by gas chromatography/electron ionization tandem mass spectrometry.

Rapid Commun Mass Spectrom 2019 Sep;33(17):1401-1409

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.

Rationale: 2-Hydroxyglutarate (2-hg) exists as enantiomers and can readily undergo cyclization to its lactone. Gas chromatography/electron ionization mass spectrometry (GC/EI-MS) has been used to separate 2-hg enantiomers in bodily fluids but the assay cannot simultaneously measure cyclic and acylic 2-hg enantiomers. Furthermore, the assignment of ion structures was not verified by complementary MS data.

Methods: GC/EI-MS and product ion analysis were used to obtain MS and MS/MS spectra of 2-hg, deuterated and C-labeled 2-hg, and 2-hg lactone. Ion structures and EI fragmentation mechanisms were determined by fragmentation pattern and isotopologue comparisons. Using the EI data, a GC/MS/MS assay was developed to separate and detect 2-hg enantiomers and 2-hg lactone enantiomers in blood and urine using a cyclodextrin capillary column.

Results: A new ion structure was predicted for the 85 m/z fragment than what was previously hypothesized, and the 117 m/z ion was the only fragment unique to the linear 2-hg compound. MS/MS data suggested that the majority of the fragments were the result of secondary fragmentation. Finally, separation of serum and urine 2-hg and 2-hg lactone enantiomers was achieved, and the acyclic 2-hg compound was found to be the major compound detected, though the amount of lactone detected was considerable in a number of samples.

Conclusions: Unique EI fragmentation pathways for both 2-hg and the 2-hg lactone have been described. Subsequently, the GC/MS/MS assay presented herein has significant potential as a novel clinical assay as it separates and detects both 2-hg enantiomers and the 2-hg lactone enantiomers, a capability which has not been previously demonstrated by any other assay to date.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rcm.8485DOI Listing
September 2019

Measurement of Postreplicative DNA Metabolism and Damage in the Rodent Brain.

Chem Res Toxicol 2015 Dec 24;28(12):2352-63. Epub 2015 Nov 24.

Department of Pharmacology and Toxicology, ‡Department of Biochemistry and Molecular Biology, §Department of Anesthesiology, ∥Department of Internal Medicine, Division of Hematology and Oncology, ⊥Moody Project for Translational Traumatic Brain Injury Research, Department of Anesthesiology, University of Texas Medical Branch , Galveston, Texas 77555, United States.

The DNA of all organisms is metabolically active due to persistent endogenous DNA damage, repair, and enzyme-mediated base modification pathways important for epigenetic reprogramming and antibody diversity. The free bases released from DNA either spontaneously or by base excision repair pathways constitute DNA metabolites in living tissues. In this study, we have synthesized and characterized the stable-isotope standards for a series of pyrimidines derived from the normal DNA bases by oxidation and deamination. We have used these standards to measure free bases in small molecule extracts from rat brain. Free bases are observed in extracts, consistent with both endogenous DNA damage and 5-methylcytosine demethylation pathways. The most abundant free base observed is uracil, and the potential sources of uracil are discussed. The free bases measured in tissue extracts constitute the end product of DNA metabolism and could be used to reveal metabolic disturbances in human disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.chemrestox.5b00359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986959PMC
December 2015

ESI-MS/MS and MALDI-IMS Localization Reveal Alterations in Phosphatidic Acid, Diacylglycerol, and DHA in Glioma Stem Cell Xenografts.

J Proteome Res 2015 Jun 29;14(6):2511-9. Epub 2015 Apr 29.

§UTMB Cancer Center, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1074, United States.

Glioblastoma (GBM) is the most common adult primary brain tumor. Despite aggressive multimodal therapy, the survival of patients with GBM remains dismal. However, recent evidence has demonstrated the promise of bone marrow-derived mesenchymal stem cells (BM-hMSCs) as a therapeutic delivery vehicle for anti-glioma agents due to their ability to migrate or home to human gliomas. While several studies have demonstrated the feasibility of harnessing the homing capacity of BM-hMSCs for targeted delivery of cancer therapeutics, it is now also evident, based on clinically relevant glioma stem cell (GSC) models of GBMs, that BM-hMSCs demonstrate variable tropism toward these tumors. In this study, we compared the lipid environment of GSC xenografts that attract BM-hMSCs (N = 9) with those that do not attract (N = 9) to identify lipid modalities that are conducive to homing of BM-hMSC to GBMs. We identified lipids directly from tissue by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) of lipid extracts. Several species of signaling lipids, including phosphatidic acid (PA 36:2, PA 40:5, PA 42:5, and PA 42:7) and diacylglycerol (DAG 34:0, DAG 34:1, DAG 36:1, DAG 38:4, DAG 38:6, and DAG 40:6), were lower in attracting xenografts. Molecular lipid images showed that PA (36:2), DAG (40:6), and docosahexaenoic acid (DHA) were decreased within tumor regions of attracting xenografts. Our results provide the first evidence for lipid signaling pathways and lipid-mediated tumor inflammatory responses in the homing of BM-hMSCs to GSC xenografts. Our studies provide new fundamental knowledge on the molecular correlates of the differential homing capacity of BM-hMSCs toward GSC xenografts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.5b00076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912016PMC
June 2015

Quantitative proteomics reveals protein-protein interactions with fibroblast growth factor 12 as a component of the voltage-gated sodium channel 1.2 (nav1.2) macromolecular complex in Mammalian brain.

Mol Cell Proteomics 2015 May 27;14(5):1288-300. Epub 2015 Feb 27.

From the ‡Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, Texas, 77555-0617;

Voltage-gated sodium channels (Nav1.1-Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein-protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.M114.040055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424400PMC
May 2015

Use of ENCODE resources to characterize novel proteoforms and missing proteins in the human proteome.

J Proteome Res 2015 Feb 26;14(2):603-8. Epub 2014 Nov 26.

Department of Pharmacology and Toxicology and ‡Biochemistry and Molecular Biology, UTMB Cancer Center, University of Texas Medical Branch , Galveston, Texas 77555, United States.

We describe the utility of integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the "missing proteins", novel proteoforms, and PTMs. On one hand, databases in combination with bioinformatic tools are efficiently utilized to establish microarray-based transcript analysis and supply rapid protein identifications in clinical samples. On the other hand, sequence libraries are the foundation of targeted protein identification and quantification using mass spectrometric and immunoaffinity techniques. The results from combining proteoENCODEdb searches with experimental mass spectral data indicate that some alternative splicing forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr500564qDOI Listing
February 2015

Integrative biological analysis for neuropsychopharmacology.

Neuropsychopharmacology 2014 Jan 26;39(1):5-23. Epub 2013 Jun 26.

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.

Although advances in psychotherapy have been made in recent years, drug discovery for brain diseases such as schizophrenia and mood disorders has stagnated. The need for new biomarkers and validated therapeutic targets in the field of neuropsychopharmacology is widely unmet. The brain is the most complex part of human anatomy from the standpoint of number and types of cells, their interconnections, and circuitry. To better meet patient needs, improved methods to approach brain studies by understanding functional networks that interact with the genome are being developed. The integrated biological approaches--proteomics, transcriptomics, metabolomics, and glycomics--have a strong record in several areas of biomedicine, including neurochemistry and neuro-oncology. Published applications of an integrated approach to projects of neurological, psychiatric, and pharmacological natures are still few but show promise to provide deep biological knowledge derived from cells, animal models, and clinical materials. Future studies that yield insights based on integrated analyses promise to deliver new therapeutic targets and biomarkers for personalized medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/npp.2013.156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3857644PMC
January 2014

Polar aprotic modifiers for chromatographic separation and back-exchange reduction for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

J Am Soc Mass Spectrom 2012 Apr;23(4):699-707

Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, FL 32306, USA.

Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein–protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) and supercritical fluid chromatography have been shown to decrease back-exchange. Here, we show that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13361-011-0329-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835171PMC
April 2012

Epitope mapping of a 95 kDa antigen in complex with antibody by solution-phase amide backbone hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

Anal Chem 2011 Sep 23;83(18):7129-36. Epub 2011 Aug 23.

Department Chemistry & Biochemistry, Florida State University, Tallahassee, Florida 32306, United States.

The epitopes of a homohexameric food allergen protein, cashew Ana o 2, identified by two monoclonal antibodies, 2B5 and 1F5, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and the results were compared to previous mapping by immunological and mutational analyses. Antibody 2B5 defines a conformational epitope, and 1F5 defines a linear epitope. Intact murine IgG antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen-monoclonal antibody (Ag-mAb) complexes. mAb-complexed and uncomplexed (free) rAna o 2 were then subjected to HDX. HDX instrumentation and automation were optimized to achieve high sequence coverage by protease XIII digestion. The regions protected from H/D exchange upon antibody binding overlap and thus confirm the previously identified epitope-bearing segments: the first extension of HDX monitored by mass spectrometry to a full-length antigen-antibody complex in solution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ac201501zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173601PMC
September 2011

Characterization of the phosphoproteome in androgen-repressed human prostate cancer cells by Fourier transform ion cyclotron resonance mass spectrometry.

J Proteome Res 2011 Sep 26;10(9):3920-8. Epub 2011 Jul 26.

Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, Florida 32306, United States.

Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO(2)-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO(2) approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC-MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr2000144DOI Listing
September 2011

Complexation and Calcium-Induced Conformational Changes in the Cardiac Troponin Complex Monitored by Hydrogen/Deuterium Exchange and FT-ICR Mass Spectrometry.

Int J Mass Spectrom 2011 Apr;302(1-3):116-124

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306.

Cardiac muscle contraction is regulated by the heterotrimeric complex: troponin. We apply solution-phase hydrogen/deuterium exchange monitored by FT-ICR mass spectrometry to study the structural dynamics and the Ca-induced conformational changes of the cardiac isoform of troponin, by comparing H/D exchange rate constants for TnC alone, the binary TnC:TnI complex, and the ternary TnC:TnI:TnT complex for Ca-free and Ca-saturated states. The wide range of exchange rate constants indicates that the complexes possess both highly flexible and very rigid domains. Fast exchange rates were observed for the N-terminal extension of TnI (specific to the cardiac isoform), the DE linker in TnC alone, and the mobile domain of TnI. The slowest rates were for the IT coiled-coil that grants stability and stiffness to the complex. Ca(2+) binding to site II of the N-lobe of TnC induces short-range allosteric effects, mainly protection for the C-lobe of TnC that transmits long-range conformational changes that reach the IT coiled-coil and even TnT1. The present results corroborate prior X-ray crystallography and NMR interpretations and also illuminate domains that were not resolved or truncated in those experiments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijms.2010.08.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3134279PMC
April 2011

Determining and interpreting correlations in lipidomic networks found in glioblastoma cells.

BMC Syst Biol 2010 Sep 7;4:126. Epub 2010 Sep 7.

Department of Computer Science, Karlsruhe Institute of Technology, Karlsruhe D-76128, Germany.

Background: Intelligent and multitiered quantitative analysis of biological systems rapidly evolves to a key technique in studying biomolecular cancer aspects. Newly emerging advances in both measurement as well as bio-inspired computational techniques have facilitated the development of lipidomics technologies and offer an excellent opportunity to understand regulation at the molecular level in many diseases.

Results: We present computational approaches to study the response of glioblastoma U87 cells to gene- and chemo-therapy. To identify distinct biomarkers and differences in therapeutic outcomes, we develop a novel technique based on graph-clustering. This technique facilitates the exploration and visualization of co-regulations in glioblastoma lipid profiling data. We investigate the changes in the correlation networks for different therapies and study the success of novel gene therapies targeting aggressive glioblastoma.

Conclusions: The novel computational paradigm provides unique "fingerprints" by revealing the intricate interactions at the lipidome level in glioblastoma U87 cells with induced apoptosis (programmed cell death) and thus opens a new window to biomedical frontiers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1752-0509-4-126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944140PMC
September 2010

New reagents for enhanced liquid chromatographic separation and charging of intact protein ions for electrospray ionization mass spectrometry.

Anal Chem 2010 Sep;82(17):7515-9

Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, USA.

Electrospray ionization produces multiply charged ions, thereby lowering the mass-to-charge ratio for peptides and small proteins to a range readily accessed by quadrupole ion trap, orbitrap, and ion cyclotron resonance (ICR) mass analyzers (m/z = 400-2000). For Fourier transform mass analyzers (orbitrap and ICR), higher charge also improves signal-to-noise ratio, mass resolution, and mass accuracy. Addition of m-nitrobenzyl alcohol (m-NBA) or sulfolane has previously been shown to increase the charge states of proteins. Moreover, polar aprotic dimethylformamide (DMF) improves chromatographic separation of proteolytic peptides for mass analysis of solution-phase protein hydrogen/deuterium exchange for improved (78-96%) sequence coverage. Here, we show that addition of each of the various modifiers (DMF, thiodiglycol, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone) can significantly increase the charge states of proteins up to 78 kDa. Moreover, incorporation of the same modifiers into reversed-phase liquid chromatography solvents improves sensitivity, charging, and chromatographic resolution for intact proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ac1016858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2932825PMC
September 2010

Computing H/D-exchange rates of single residues from data of proteolytic fragments.

BMC Bioinformatics 2010 Aug 11;11:424. Epub 2010 Aug 11.

Institut für Informatik, Fachbereich 08, Staudingerweg 9, 55099 Mainz, Germany.

Background: Protein conformation and protein/protein interaction can be elucidated by solution-phase Hydrogen/Deuterium exchange (sHDX) coupled to high-resolution mass analysis of the digested protein or protein complex. In sHDX experiments mutant proteins are compared to wild-type proteins or a ligand is added to the protein and compared to the wild-type protein (or mutant). The number of deuteriums incorporated into the polypeptides generated from the protease digest of the protein is related to the solvent accessibility of amide protons within the original protein construct.

Results: In this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based on combinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX data of overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residue resolution.

Conclusions: With our new method it is possible to automatically determine deuterium exchange with higher spatial resolution than the level of digested fragments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2105-11-424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2936929PMC
August 2010

Sites and extent of selenomethionine incorporation into recombinant Cas6 protein by top-down and bottom-up proteomics with 14.5 T Fourier transform ion cyclotron resonance mass spectrometry.

Rapid Commun Mass Spectrom 2010 Aug;24(16):2386-92

Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, Florida 32306, USA.

Selenomethionine-modified proteins can improve X-ray crystallographic structural resolution by multi-wavelength anomalous diffraction (MAD) phasing. However, the specificity and extent of selenomethionine incorporation must first be assessed. Bottom-up and top-down proteomics with a modified 14.5 T LTQ Fourier transform ion cyclotron resonance mass spectrometer offer a quick, accurate, and robust method to locate and quantify selenomethionine incorporation after auxotrophic expression. Selenomethionine (methionine with sulfur replaced by selenium) has a different natural-abundance isotopic distribution and a mass increase of 47.94 Da relative to wild-type methionine. Here, both wild-type and selenomethionine-substituted forms of the Cas6 protein containing 'clustered regularly interspaced short palindromic repeats' (CRISPRs) were expressed and purified. Comparative bottom-up and top-down proteomics confirmed that all six methionines were fully replaced by selenomethionines in Se-Cas6.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/rcm.4655DOI Listing
August 2010

Overexpression of ST6GalNAcV, a ganglioside-specific alpha2,6-sialyltransferase, inhibits glioma growth in vivo.

Proc Natl Acad Sci U S A 2010 Jul 28;107(28):12646-51. Epub 2010 Jun 28.

The Falk Center for Molecular Therapeutics, Department of Biomedical Engineering, Northwestern University, Evanston, IL 60201, USA.

Aberrant cell-surface glycosylation patterns are present on virtually all tumors and have been linked to tumor progression, metastasis, and invasivity. We have shown that expressing a normally quiescent, glycoprotein-specific alpha2,6-sialyltransferase (ST6Gal1) gene in gliomas inhibited invasivity in vitro and tumor formation in vivo. To identify other glycogene targets with therapeutic potential, we created a focused 45-mer oligonucleotide microarray platform representing all of the cloned human glycotranscriptome and examined the glycogene expression profiles of 10 normal human brain specimens, 10 malignant gliomas, and 7 human glioma cell lines. Among the many significant changes in glycogene expression observed, of particular interest was the observation that an additional alpha2,6-sialyltransferase, ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha2,6-sialyltransferase 5 (ST6GalNAcV), was expressed at very low levels in all glioma and glioma cell lines examined compared with normal brain. ST6GalNAcV catalyzes the formation of the terminal alpha2,6-sialic acid linkages on gangliosides. Stable transfection of ST6GalNAcV into U373MG glioma cells produced (i) no change in alpha2,6-linked sialic acid-containing glycoproteins, (ii) increased expression of GM2alpha and GM3 gangliosides and decreased expression of GM1b, Gb3, and Gb4, (iii) marked inhibition of in vitro invasivity, (iv) modified cellular adhesion to fibronectin and laminin, (v) increased adhesion-mediated protein tyrosine phosphorylation of HSPA8, and (vi) inhibition of tumor growth in vivo. These results strongly suggest that modulation of the synthesis of specific glioma cell-surface glycosphingolipids alters invasivity in a manner that may have significant therapeutic potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0909862107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906591PMC
July 2010

Liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric characterization of N-linked glycans and glycopeptides.

Anal Chem 2010 Aug;82(15):6542-8

Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, Florida 32306, USA.

We combine liquid chromatography, electrospray ionization, and Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS) to determine the sugar composition, linkage pattern, and attachment sites of N-linked glycans. N-linked glycans were enzymatically released from glycoproteins with peptide N-glycosidase F, followed by purification with graphitized carbon cartridge solid-phase extraction and separation over a TSK-Gel Amide80 column under hydrophilic interaction chromatography (HILIC) conditions. Unique glycopeptide compositions were determined from experimentally measured masses for different combinations of glycans and glycopeptides. The method was validated by identifying four peptides glycosylated so as to yield 12 glycopeptides unique in glycan composition for the standard glycoprotein, bovine alpha-2-HS-glycoprotein. We then assigned a total of 137 unique glycopeptide compositions from 18 glycoproteins from fetal bovine serum, and the glycan structures for most of the assigned glycopeptides were heterogeneous. Highly accurate FT-ICR mass measurement is essential for reliable identification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ac1008833DOI Listing
August 2010

Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry.

Anal Chem 2010 Apr;82(8):3293-9

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA.

Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein-protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of (13)C and/or (15)N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ((12)C(c)(1)H(h)(14)N(n)(16)O(o)(32)S(s)), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ac100079zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878611PMC
April 2010

Glycomic and transcriptomic response of GSC11 glioblastoma stem cells to STAT3 phosphorylation inhibition and serum-induced differentiation.

J Proteome Res 2010 May;9(5):2098-108

Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310, USA.

A glioblastoma stem cell (GSC) line, GSC11, grows as neurospheres in serum-free media supplemented with EGF (epidermal growth factor) and bFGF (basic fibroblast growth factor), and, if implanted in nude mice brains, will recapitulate high-grade glial tumors. Treatment with a STAT3 (signal transducer and activator of transcription 3) phosphorylation inhibitor (WP1193) or 10% FBS (fetal bovine serum) both led to a decrease in expression of the stem cell marker CD133 in GSC11 cells, but differed in phenotype changes. Altered glycolipid profiles were associated with some differentially expressed glycogenes. In serum treated cells, an overall increase in glycosphingolipids may be due to increased expression of ST6GALNAC2, a sialyltransferase. Serum treated cells express more phosphatidylcholine (PC), short chain sphingomyelin (SM) and unsaturated long chain phosphatidylinositol (PI). Decrease of a few glycosphingolipids in the STAT3 phosphorylation inhibited cells may be linked to decreased transcripts of ST6GALNAC2 and UGCGL2, a glucosylceramide synthase. A rare 3-sulfoglucuronylparagloboside carrying HNK1 (human natural killer-1) epitope was found expressed in the GSC11 and the phenotypically differentiated cells. Its up-regulation correlates with increased transcripts of a HNK1 biosynthesis gene, B3GAT2 after serum treatment. Taken together with a quantitative phosphoproteomic study of the same GSC line (C. L. Nilsson, et al. J. Proteome Res. 2010, 9, 430-443), this report represents the most complete systems biology study of cancer stem cell (CSC) differentiation to date. The synergies derived by the combination of glycomic, transcriptomic and phosphoproteomic data may aid our understanding of intracellular and cell-surface events associated with CSC differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr900793aDOI Listing
May 2010

Automated data reduction for hydrogen/deuterium exchange experiments, enabled by high-resolution Fourier transform ion cyclotron resonance mass spectrometry.

J Am Soc Mass Spectrom 2010 Apr 4;21(4):550-8. Epub 2010 Jan 4.

Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL 32310-4005, USA.

Mass analysis of proteolytic fragment peptides following hydrogen/deuterium exchange offers a general measure of solvent accessibility/hydrogen bonding (and thus conformation) of solution-phase proteins and their complexes. The primary problem in such mass analyses is reliable and rapid assignment of mass spectral peaks to the correct charge state and degree of deuteration of each fragment peptide, in the presence of substantial overlap between isotopic distributions of target peptides, autolysis products, and other interferant species. Here, we show that at sufficiently high mass resolving power (m/Delta m(50%) > or = 100,000), it becomes possible to resolve enough of those overlaps so that automated data reduction becomes possible, based on the actual elemental composition of each peptide without the need to deconvolve isotopic distributions. We demonstrate automated, rapid, reliable assignment of peptide masses from H/D exchange experiments, based on electrospray ionization FT-ICR mass spectra from H/D exchange of solution-phase myoglobin. Combined with previously demonstrated automated data acquisition for such experiments, the present data reduction algorithm enhances automation (and thus expands generality and applicability) for high-resolution mass spectrometry-based analysis of H/D exchange of solution-phase proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jasms.2009.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901854PMC
April 2010

Conformational states of human purine nucleoside phosphorylase at rest, at work, and with transition state analogues.

Biochemistry 2010 Mar;49(9):2058-67

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; K(d) = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; K(d) = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP.PO(4).inosine <--> PNP.Hx.R-1-P) and inhibited complexes (PNP.PO(4).DATMe-ImmH and PNP.PO(4).ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP.PO(4)) in extended incubations. The PNP.PO(4).ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi902041jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832846PMC
March 2010

Simultaneous reduction and digestion of proteins with disulfide bonds for hydrogen/deuterium exchange monitored by mass spectrometry.

Anal Chem 2010 Feb;82(4):1450-4

Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, USA.

Proteolyzed peptides provide the basis for mass-analyzed hydrogen/deuterium exchange (HDX) for mapping solvent access to various segments of solution-phase proteins. Aspergillus saitoi protease type XIII and porcine pepsin can generate peptides of overlapping sequences and high sequence coverage. However, if disulfide bonds are present, proteolysis can be severely limited, particularly in the vicinity of the disulfide linkage(s). Disulfide bonds cannot be reduced before or during the H/D exchange reaction without affecting the protein higher-order structure. Here, we demonstrate simultaneous quench/digestion/reduction following H/D exchange, for subsequent mass analysis. Proteolysis is conducted in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) and urea, and all other steps of the H/D exchange and analysis are maintained. This method yields dramatically increased sequence coverage and localization of solvent-exposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ac902550nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825376PMC
February 2010

Drug binding and resistance mechanism of KIT tyrosine kinase revealed by hydrogen/deuterium exchange FTICR mass spectrometry.

Protein Sci 2010 Apr;19(4):703-15

Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310-4005, USA.

Mutations of the receptor tyrosine kinase KIT are linked to certain cancers such as gastrointestinal stromal tumors (GISTs). Biophysical, biochemical, and structural studies have provided insight into the molecular basis of resistance to the KIT inhibitors, imatinib and sunitinib. Here, solution-phase hydrogen/deuterium exchange (HDX) and direct binding mass spectrometry experiments provide a link between static structure models and the dynamic equilibrium of the multiple states of KIT, supporting that sunitinib targets the autoinhibited conformation of WT-KIT. The D816H mutation shifts the KIT conformational equilibrium toward the activated state. The V560D mutant exhibits two low energy conformations: one is more flexible and resembles the D816H mutant shifted toward the activated conformation, and the other is less flexible and resembles the wild-type KIT in the autoinhibited conformation. This result correlates with the V560D mutant exhibiting a sensitivity to sunitinib that is less than for WT KIT but greater than for KIT D816H. These findings support the elucidation of the resistance mechanism for the KIT mutants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2867011PMC
April 2010

Sequential proteolysis and high-field FTICR MS to determine disulfide connectivity and 4-maleimide TEMPO spin-label location in L126C GM2 activator protein.

Anal Chem 2009 Sep;81(18):7611-7

Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, USA.

The GM2 activator protein (GM2AP) is an 18 kDa nonenzymatic accessory protein involved in the degradation of neuronal gangliosides. Genetic mutations of GM2AP can disrupt ganglioside catabolism and lead to deadly lysosomal storage disorders. Crystallography of wild-type GM2AP reveals 4 disulfide bonds and multiple conformations of a flexible loop region that is thought to be involved in lipid binding. To extend the crystallography results, a cysteine construct (L126C) was expressed and modified with 4-maleimide TEMPO for electron paramagnetic resonance (EPR) studies. However, because a ninth cysteine has been added by site-directed mutagenesis and the protein was expressed in E. coli in the form of inclusion bodies, the protein could misfold during expression. To verify correct protein folding and labeling, a sequential multiple-protease digestion, nano-liquid chromatograph (LC) electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry assay was developed. High-magnetic field and robust automatic gain control results in subppm mass accuracy for location of the spin-labeled cysteine and verification of proper connectivity of the four disulfide bonds. The sequential multiple protease digestion strategy and ultrahigh mass accuracy provided by FTICR MS allow for rapid and unequivocal assignment of relevant peptides and provide a simple pipeline for analyzing other GM2AP constructs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ac9009935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757923PMC
September 2009

Global stability analysis and robust design of multi-time-scale biological networks under parametric uncertainties.

Neural Netw 2009 Jul-Aug;22(5-6):658-63. Epub 2009 Jul 14.

Department of Scientific Computing, Florida State University, Tallahassee, FL 32310-6046, United States.

Biological networks are prone to internal parametric fluctuations and external noises. Robustness represents a crucial property of these networks, which militates the effects of internal fluctuations and external noises. In this paper biological networks are formulated as coupled nonlinear differential systems operating at different time-scales under vanishing perturbations. In contrast to previous work viewing biological parametric uncertain systems as perturbations to a known nominal linear system, the perturbed biological system is modeled as nonlinear perturbations to a known nonlinear idealized system and is represented by two time-scales (subsystems). In addition, conditions for the existence of a global uniform attractor of the perturbed biological system are presented. By using an appropriate Lyapunov function for the coupled system, a maximal upper bound for the fast time-scale associated with the fast state is derived. The proposed robust system design principles are potentially applicable to robust biosynthetic network design. Finally, two examples of two important biological networks, a neural network and a gene regulatory network, are presented to illustrate the applicability of the developed theoretical framework.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neunet.2009.06.051DOI Listing
November 2009
-->