42 results on '"Timothy J. Waybright"'
Search Results
2. Supplementary_Figure_Legends from mTOR Inhibition Mitigates Enhanced mRNA Translation Associated with the Metastatic Phenotype of Osteosarcoma Cells In Vivo
- Author
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Chand Khanna, Peter C. Scacheri, Timothy M. Fan, Ming Zhou, Daniel L. Gustafson, Ryan J. Hansen, Timothy J. Waybright, Ling Ren, Michael M. Lizardo, Allyson Koyen, Arnulfo Mendoza, and James J. Morrow
- Abstract
Legends for Supplementary Tables and Figures
- Published
- 2023
3. Data from mTOR Inhibition Mitigates Enhanced mRNA Translation Associated with the Metastatic Phenotype of Osteosarcoma Cells In Vivo
- Author
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Chand Khanna, Peter C. Scacheri, Timothy M. Fan, Ming Zhou, Daniel L. Gustafson, Ryan J. Hansen, Timothy J. Waybright, Ling Ren, Michael M. Lizardo, Allyson Koyen, Arnulfo Mendoza, and James J. Morrow
- Abstract
Purpose: To successfully metastasize, tumor cells must respond appropriately to biological stressors encountered during metastatic progression. We sought to test the hypothesis that enhanced efficiency of mRNA translation during periods of metastatic stress is required for metastatic competence of osteosarcoma and that this metastasis-specific adaptation is amenable to therapeutic intervention.Experimental Design: We employ novel reporter and proteomic systems that enable tracking of mRNA translation efficiency and output in metastatic osteosarcoma cells as they colonize the lungs. We test the potential to target mRNA translation as an antimetastatic therapeutic strategy through pharmacokinetic studies and preclinical assessment of the prototypic mTOR inhibitor, rapamycin, across multiple models of metastasis.Results: Metastatic osteosarcoma cells translate mRNA more efficiently than nonmetastatic cells during critical stressful periods of metastatic colonization of the lung. Rapamycin inhibits translational output during periods of metastatic stress, mitigates lung colonization, and prolongs survival. mTOR-inhibiting exposures of rapamycin are achievable in mice using treatment schedules that correspond to human doses well below the MTDs defined in human patients, and as such are very likely to be tolerated over long exposures alone and in combination with other agents.Conclusions: Metastatic competence of osteosarcoma cells is dependent on efficient mRNA translation during stressful periods of metastatic progression, and the mTOR inhibitor, rapamycin, can mitigate this translation and inhibit metastasis in vivo. Our data suggest that mTOR pathway inhibitors should be reconsidered in the clinic using rationally designed dosing schedules and clinical metrics related to metastatic progression. Clin Cancer Res; 22(24); 6129–41. ©2016 AACR.
- Published
- 2023
4. Supplementary Table 1 from mTOR Inhibition Mitigates Enhanced mRNA Translation Associated with the Metastatic Phenotype of Osteosarcoma Cells In Vivo
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Chand Khanna, Peter C. Scacheri, Timothy M. Fan, Ming Zhou, Daniel L. Gustafson, Ryan J. Hansen, Timothy J. Waybright, Ling Ren, Michael M. Lizardo, Allyson Koyen, Arnulfo Mendoza, and James J. Morrow
- Abstract
Summary of BONCAT proteomic analysis of mRNA translation during metastasis
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- 2023
5. Supplementary Figure 2 from mTOR Inhibition Mitigates Enhanced mRNA Translation Associated with the Metastatic Phenotype of Osteosarcoma Cells In Vivo
- Author
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Chand Khanna, Peter C. Scacheri, Timothy M. Fan, Ming Zhou, Daniel L. Gustafson, Ryan J. Hansen, Timothy J. Waybright, Ling Ren, Michael M. Lizardo, Allyson Koyen, Arnulfo Mendoza, and James J. Morrow
- Abstract
Highly metastatic osteosarcoma cells activate the mTOR pathway during metastatic progression
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- 2023
6. Supplementary Figure 1 from mTOR Inhibition Mitigates Enhanced mRNA Translation Associated with the Metastatic Phenotype of Osteosarcoma Cells In Vivo
- Author
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Chand Khanna, Peter C. Scacheri, Timothy M. Fan, Ming Zhou, Daniel L. Gustafson, Ryan J. Hansen, Timothy J. Waybright, Ling Ren, Michael M. Lizardo, Allyson Koyen, Arnulfo Mendoza, and James J. Morrow
- Abstract
5' UTR structures of BONCAT identified proteins
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- 2023
7. Allosteric regulation of switch-II controls K-Ras oncogenicity
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Moon Hee Yang, Timothy H. Tran, Bethany Hunt, Rebecca Agnor, Christian W. Johnson, Timothy J. Waybright, Jonathan A. Nowak, Andrew G. Stephen, Dhirendra K. Simanshu, and Kevin M. Haigis
- Abstract
Ras proteins are GTPases that regulate a wide range of cellular processes. The activity of Ras is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Previously, we demonstrated that mutation of lysine 104 to glutamine (K104Q) attenuates the transforming capacity of oncogenic K-Ras by interrupting GEF induced nucleotide exchange. To assess the effect of this mutation in vivo, we used CRISPR/Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional K-RasLSL-G12D alleles. Consistent with our previous findings from in vitro studies, the oncogenic activity of K-RasG12D was significantly attenuated by mutation at K104 in vivo. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant K-Ras and suggest that modification at K104, for example acetylation, may also regulate its activity. In addition, animals homozygous for K104Q were viable, fertile, and arose at Mendelian frequency, indicating that K104Q is not a complete loss of function mutation. Using biochemical and structural analysis, we found that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic K-Ras. Finally, we discovered an allosteric regulatory network consisting of K104 and residues including G75 on switch II (SWII) that is the key for regulating the stability of the α helix on SWII. In this allosteric network, K104-G75 interaction might be primary for keeping stabilization of SWII. Given the high frequency of KRAS mutations in human cancers, modulation of this network may provide a unique therapeutic approach.
- Published
- 2022
8. GAP positions catalytic h-Ras residue Q61 for GTP hydrolysis in MD simulations
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Lara A. Patel, Timothy J. Waybright, Andrew G. Stephen, and Chris Neale
- Subjects
Biophysics - Published
- 2022
9. Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein
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Simon Messing, Andrew G. Stephen, Peter Frank, William K. Gillette, Constance Agamasu, Timothy J. Waybright, and Shelley Perkins
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General Chemical Engineering ,Ion chromatography ,Protein Prenylation ,Methylation ,environment and public health ,Mass Spectrometry ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,law.invention ,Proto-Oncogene Proteins p21(ras) ,Prenylation ,law ,Animals ,Nucleotide ,Surface plasmon resonance ,chemistry.chemical_classification ,Liposome ,General Immunology and Microbiology ,Chemistry ,General Neuroscience ,Reproducibility of Results ,Surface Plasmon Resonance ,Chromatography, Ion Exchange ,Recombinant Proteins ,Membrane ,Biochemistry ,Liposomes ,Recombinant DNA ,Protein prenylation ,lipids (amino acids, peptides, and proteins) ,Baculoviridae - Abstract
Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a 'CAAX' box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.
- Published
- 2020
10. Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer
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Sriram Subramaniam, Debsindhu Bhowmik, Hugh O'Neill, Matthew Drew, Simon Messing, Dominic Esposito, William K. Gillette, Mukul Sherekar, Christopher B. Stanley, Arvind Ramanathan, Sae-Won Han, Puneet Juneja, Frank McCormick, Dwight V. Nissley, Timothy J. Waybright, Dana Rabara, and Rodolfo Ghirlando
- Subjects
0301 basic medicine ,GTPase Kras (KRAS) ,GTPase ,GTPase-activating protein (GAP) ,Medical and Health Sciences ,Biochemistry ,law.invention ,0302 clinical medicine ,law ,mitogen-activated protein kinase pathway ,Neurofibromatosis type I ,0303 health sciences ,Neurofibromin 1 ,dimerization ,biology ,Chemistry ,Biological Sciences ,Cell biology ,ras GTPase-Activating Proteins ,030220 oncology & carcinogenesis ,Protein Structure and Folding ,Ras protein ,GTPase Kras ,Cell signaling ,Biochemistry & Molecular Biology ,congenital, hereditary, and neonatal diseases and abnormalities ,Protein domain ,RASopathy ,03 medical and health sciences ,Structure-Activity Relationship ,Protein Domains ,medicine ,Humans ,cell signaling ,cancer ,Protein kinase A ,Molecular Biology ,030304 developmental biology ,GTPase-activating protein ,030102 biochemistry & molecular biology ,Cell Biology ,neurofibromin ,medicine.disease ,nervous system diseases ,030104 developmental biology ,HEK293 Cells ,NF1 ,Chemical Sciences ,biology.protein ,Suppressor ,Protein Multimerization - Abstract
Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.
- Published
- 2019
11. Abstract 2586: Identification of cancer-associated RhoA mutants and analogous K-Ras mutants that induce gain-of function despite low GTP binding
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Beatriz Sánchez-Solana, Dominic Esposito, Marian E. Durkin, Andrew G. Stephen, Douglas R. Lowy, Dunrui Wang, Brajendra K. Tripathi, Alex G. Papageorge, Yucheng Gao, Timothy J. Waybright, and Xiaolan Qian
- Subjects
Cancer Research ,RHOA ,GTP' ,biology ,Chemistry ,Mutant ,Cancer ,medicine.disease ,Cell biology ,Gain of function ,Oncology ,medicine ,biology.protein ,Identification (biology) - Abstract
While the Ras GTPases are frequently mutated, especially at codons 12, 13, or 61, the structurally related Rho GTPases, although they are implicated in tumor invasion and metastasis, are rarely mutated in solid tumors, except for diffuse gastric cancer. The vast majority of cancer-associated RHO mutations are not located in codons 14, 15, or 63, which correspond to the 3 canonical mutant Ras codons. Here we have evaluated the biological and biochemical phenotypes of several cancer-associated RhoA mutants, which indicated that some RhoA mutants have unusual phenotypes and therefore prompted us to generalize our findings to other small GTPases by constructing and analyzing the analogous mutants in K-Ras. We constructed 8 cancer-associated RhoA mutants from the TCGA database, and found that each was gain-of-function biologically. Using the Rhotekin binding assay to infer RhoGTP levels, 5 mutants (Y42C, L57V, D59G, A61D, D76G), had higher binding than WT RhoA. However, 3 mutants (G62E. L69P and L69R) did not bind Rhotekin, but did bind strongly to Citron, a Rho-dependent kinase implicated in cytokinesis and cell cycle regulation. Because the three mutants were highly transforming, we directly determined GTP/GDP binding levels, the “gold-standard” assay, for all mutants, which indicated G62E had high GTP-binding and low GDP-binding, in contrast to its negative binding to Rhotekin, while L69P and L69R had high GDP-binding and low GTP-binding similar to WT. Purified GDP-bound L69P and L69R formed a complex with Citron more efficiently than did WT GTP-bound or GDP-bound RhoA. Endogenous Citron and CDK1 contribute to anchorage-independent growth, as siRNA knockdown of either gene in lines transformed by G62E, L69R or L69P inhibited this phenotype. The cancer-associated K-Ras mutants G60E and M67L (which correspond to RhoA G62E and L69R) and also K-Ras M67R displayed gain-of-function biologically, and the GTP/GDP-binding phenotype of each was analogous to the respective RhoA mutant: high GTP-binding for G60E, and low Raf-RBD binding and high GDP-binding for M67L and M67R. The low GTP K-Ras mutants bound more strongly to the RBD domain of Ras effectors other than Raf, resulting in activation of RAP and AKT in transfected cells. Our microarray data revealed that these GDP-bound mutant K-Ras or RhoA can up-regulate cell-cycle progression genes and down-regulate tumor suppressor genes compared to WT, which may account for their transforming phenotype. We conclude that some cancer-associated RhoA mutants are transforming when GDP-bound, a phenotype that has not been described for any small GTPase. The equivalent K-Ras mutants display a similar phenotype, which implies the findings should be extrapolatable to many small GTPases. Such mutants can be strongly transforming, although they only bind a subset of preferred downstream effectors and activate their respective pathways. In addition, the widely used Rhotekin binding assay to infer RhoGTP levels may give misleading results for some mutants. Citation Format: Xiaolan Qian, Alex Papageorge, Dunrui Wang, Brajendra Tripathi, Yucheng Gao, Marian Durkin, Beatriz Sanchez-Solana, Dominic Esposito, Timothy Waybright, Andrew Stephen, Douglas R. Lowy. Identification of cancer-associated RhoA mutants and analogous K-Ras mutants that induce gain-of function despite low GTP binding [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2586.
- Published
- 2020
12. Abstract A02: Probing amino acids residues chemical reactivity of KRAS 4b using N-hydroxysuccinimide esters
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Timothy J. Waybright, Oleg Chertov, Andrew G. Stephen, and Xiaoying Ye
- Subjects
chemistry.chemical_classification ,Cancer Research ,Chemistry ,Chemical modification ,Peptide ,Small molecule ,Amino acid ,Residue (chemistry) ,Protein structure ,Oncology ,Biochemistry ,Cancer research ,HRAS ,Binding site ,Molecular Biology - Abstract
RAS oncoproteins are frequently mutated in pancreatic, colon and lung cancers and play a causal role in human carcinogenesis. Although it is a potentially good target for anticancer drugs development, it has been challenging to specifically target different forms of RAS using small molecules because of absence of evident target binding sites. Chemical reactivity of protein functional groups depends on the environment of a particular residue and reagent accessibility due to protein structure. We wanted to explore chemical reactivity of KRAS 4b functional groups using chemical reagents to identify novel sites for covalent and noncovalent drug development. NHS esters have been used for years for chemical modification of proteins. They are believed to selectively react with primary amino groups. However, there are reports in the literature that other nucleophilic groups such as sulfhydryl group of Cys can react with N-hydroxysuccinimide esters. It is possible that other functional groups may also react. Sulfo-NHS-acetate, sulfo-NHS-biotin and NHS-biotin were used for modification of K, HRAS proteins in GDP and GppNHp-loaded forms. To follow the extent of protein modification, MALDI-TOF MS was used. Generally, the reaction was performed until almost no unmodified protein was left. After modification the protein was digested by Glu-C and peptides were analyzed by MALDI-TOF MS and ES-MS/MS. A “modification index” (ratio of intensities of modified and unmodified peptide forms in MALDI-TOF MS spectra) was calculated to enable relative quantitation for all modified peptides. The comparison of the modification indexes for peptides derived from NHS-biotin modified KRAS and HRAS in GDP or GppNHp form suggested that GppNHp-loaded protein in general allows higher degree of modification. This suggests that there are inducible and expandable binding sites for small organic molecules that could be exploited for design of covalent and noncovalent drug leads. Acknowledgments: Supported by NCI contract HHSN261200800001E. Citation Format: Oleg Chertov, Xiaoying Ye, Timothy Waybright, Andrew G. Stephen. Probing amino acids residues chemical reactivity of KRAS 4b using N-hydroxysuccinimide esters [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A02.
- Published
- 2020
13. Structures of N-terminally processed KRAS provide insight into the role of N-acetylation
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Timothy J. Waybright, Andrew G. Stephen, Frank McCormick, William K. Gillette, Timothy H. Tran, Srisathiyanarayanan Dharmaiah, Wupeng Yan, Constance Agamasu, Patrick Alexander, Dominic Esposito, Dhirendra K. Simanshu, Dwight V. Nissley, and Simon Messing
- Subjects
0301 basic medicine ,Models, Molecular ,Protein Conformation ,lcsh:Medicine ,Sequence Homology ,Plasma protein binding ,medicine.disease_cause ,Crystallography, X-Ray ,0302 clinical medicine ,Protein structure ,Models ,Catalytic Domain ,2.1 Biological and endogenous factors ,Magnesium ,Aetiology ,lcsh:Science ,Peptide sequence ,Cancer ,Guanylyl Imidodiphosphate ,Multidisciplinary ,Crystallography ,Chemistry ,Acetylation ,Molecular biophysics ,Amino Acid ,KRAS ,Protein Binding ,Sequence alignment ,Guanosine Diphosphate ,Article ,Proto-Oncogene Proteins p21(ras) ,03 medical and health sciences ,Structure-Activity Relationship ,medicine ,Structure–activity relationship ,Humans ,Amino Acid Sequence ,Binding site ,Protein Processing ,X-ray crystallography ,Sequence Homology, Amino Acid ,lcsh:R ,Post-Translational ,Molecular ,Hydrogen Bonding ,030104 developmental biology ,Biophysics ,X-Ray ,lcsh:Q ,Protein Processing, Post-Translational ,Sequence Alignment ,030217 neurology & neurosurgery - Abstract
Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.
- Published
- 2018
14. Phosphorylation of centromeric histone H3 variant regulates chromosome segregation inSaccharomyces cerevisiae
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Lars Boeckmann, John S. Choy, Munira A. Basrai, Yoshimitsu Takahashi, Christopher McAndrew, Anthony R. Dawson, Christopher D. Heger, Timothy J. Waybright, Prashant K. Mishra, Timothy D. Veenstra, May Y. Szeto, Paul K. Goldsmith, Richard Baker, and Wei-Chun Au
- Subjects
inorganic chemicals ,Saccharomyces cerevisiae Proteins ,Chromosomal Proteins, Non-Histone ,Blotting, Western ,Centromere ,Molecular Sequence Data ,Mutant ,Biorientation ,Aurora B kinase ,Cell Cycle Proteins ,macromolecular substances ,Saccharomyces cerevisiae ,Biology ,environment and public health ,Chromosome segregation ,Aurora Kinases ,Tandem Mass Spectrometry ,Chromosome Segregation ,Aurora Kinase B ,Amino Acid Sequence ,Phosphorylation ,Kinetochores ,Molecular Biology ,Binding Sites ,Sequence Homology, Amino Acid ,Kinetochore ,Nuclear Functions ,Nuclear Proteins ,Articles ,Cell Biology ,Molecular biology ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,Mutation ,bacteria ,Microtubule-Associated Proteins ,Chromatography, Liquid - Abstract
Cse4 is posttranslationally modified in Saccharomyces cerevisiae. Ipl1 contributes to Cse4 phosphorylation in vivo and in vitro. Phosphorylation of Cse4 at centromeres is enhanced in response to nocodazole or reduced cohesion. The results suggest that phosphorylation of Cse4 ensures faithful chromosome segregation., The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein–labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.
- Published
- 2013
15. mTOR Inhibition Mitigates Enhanced mRNA Translation Associated with the Metastatic Phenotype of Osteosarcoma Cells In Vivo
- Author
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Allyson E. Koyen, Timothy J. Waybright, Arnulfo Mendoza, Ling Ren, Timothy M. Fan, Peter C. Scacheri, Chand Khanna, Michael M. Lizardo, Ryan J. Hansen, Daniel L. Gustafson, Ming Zhou, and James J. Morrow
- Subjects
0301 basic medicine ,Proteomics ,Cancer Research ,Mice, Nude ,Apoptosis ,Mice, SCID ,Article ,Metastasis ,03 medical and health sciences ,Mice ,0302 clinical medicine ,In vivo ,Cell Line, Tumor ,medicine ,Animals ,Humans ,RNA, Messenger ,Protein Kinase Inhibitors ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,Sirolimus ,Mice, Inbred BALB C ,Osteosarcoma ,business.industry ,TOR Serine-Threonine Kinases ,Cancer ,Translation (biology) ,medicine.disease ,Phenotype ,Xenograft Model Antitumor Assays ,030104 developmental biology ,Oncology ,Cell culture ,030220 oncology & carcinogenesis ,Immunology ,Cancer research ,Female ,business ,Signal Transduction - Abstract
Purpose: To successfully metastasize, tumor cells must respond appropriately to biological stressors encountered during metastatic progression. We sought to test the hypothesis that enhanced efficiency of mRNA translation during periods of metastatic stress is required for metastatic competence of osteosarcoma and that this metastasis-specific adaptation is amenable to therapeutic intervention. Experimental Design: We employ novel reporter and proteomic systems that enable tracking of mRNA translation efficiency and output in metastatic osteosarcoma cells as they colonize the lungs. We test the potential to target mRNA translation as an antimetastatic therapeutic strategy through pharmacokinetic studies and preclinical assessment of the prototypic mTOR inhibitor, rapamycin, across multiple models of metastasis. Results: Metastatic osteosarcoma cells translate mRNA more efficiently than nonmetastatic cells during critical stressful periods of metastatic colonization of the lung. Rapamycin inhibits translational output during periods of metastatic stress, mitigates lung colonization, and prolongs survival. mTOR-inhibiting exposures of rapamycin are achievable in mice using treatment schedules that correspond to human doses well below the MTDs defined in human patients, and as such are very likely to be tolerated over long exposures alone and in combination with other agents. Conclusions: Metastatic competence of osteosarcoma cells is dependent on efficient mRNA translation during stressful periods of metastatic progression, and the mTOR inhibitor, rapamycin, can mitigate this translation and inhibit metastasis in vivo. Our data suggest that mTOR pathway inhibitors should be reconsidered in the clinic using rationally designed dosing schedules and clinical metrics related to metastatic progression. Clin Cancer Res; 22(24); 6129–41. ©2016 AACR.
- Published
- 2016
16. Characterization of Recombinant Human IL-15 Deamidation and Its Practical Elimination through Substitution of Asparagine 77
- Author
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Jianwei Zhu, David F. Nellis, Terry L. Sumpter, Xiaoyi Yang, Angela Korrell, Roy E. Nelson, Timothy J. Waybright, Man-Shiow Jiang, Amy M. Zheng, John A. Gilly, Emily M. Pritt, Lauren E. Lucernoni, Mark Rogers, Hengguang Jiang, Jason L. Yovandich, Dennis F. Michiel, Dominic Esposito, George C. Knapp, Lauren V. Procter, George Mitra, Richard Davis, and Vinay V. Vyas
- Subjects
T-Lymphocytes ,Molecular Sequence Data ,Kinetics ,Pharmaceutical Science ,Cell Line ,law.invention ,Mice ,chemistry.chemical_compound ,law ,Animals ,Humans ,Pharmacology (medical) ,Amino Acid Sequence ,Asparagine ,Deamidation ,Cell Proliferation ,Interleukin-15 ,Pharmacology ,chemistry.chemical_classification ,Chromatography ,Ethanol ,Protein Stability ,Chemistry ,Organic Chemistry ,Protein engineering ,Recombinant Proteins ,Amino acid ,Enzyme ,Amino Acid Substitution ,Biochemistry ,Recombinant DNA ,Molecular Medicine ,Biotechnology - Abstract
The use of recombinant human interleukin (rhIL)-15 as a potential therapeutic immune modulator and anticancer agent requires pure, stable preparations. However, purified rhIL-15 preparations readily accumulated heterogeneities. We sought to improve rhIL-15 stability through process, formulation, and targeted amino acid changes. The solution state of rhIL-15 versus buffer composition and temperature was studied using SEC and IEX methods. rhIL-15 deamidation was confirmed using RP-HPLC/ESI-MS, enzymatic labeling, and peptide mapping. Deamidation kinetics were measured versus buffer composition and pH using RP-HPLC. Deamidation-resistant rhIL-15 variants (N77A, N77S, N77Q, G78A, and [N71S/N72A/N77A]) were produced in E. coli, then assayed for T-cell culture expansion potency and deamidation resistance. Adding 20% ethanol to buffers or heating at ≥32°C dispersed rhIL-15 transient pairs, improving purification efficiencies. Asparagine 77 deamidated rapidly at pH 7.4 with activation energy of 22.9 kcal per mol. Deamidation in citrate buffer was 17-fold slower at pH 5.9 than at pH 7.4. Amino acid substitutions at N77 or G78 slowed deamidation ≥23-fold. rhIL-15 variants N77A and (N71S/N72A/N77A) were active in a CTLL-2 proliferation assay equivalent to unsubstituted rhIL-15. The N77A and (N71S/N72A/N77A) rhIL-15 variants are resistant to deamidation and remain potent, thus providing enhanced drug substances for clinical evaluation.
- Published
- 2011
17. Molecular profiling of the human nasal epithelium: A proteomics approach
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Francisco M. Couto, Daniel Faria, Josip Blonder, Nuno Charro, King C. Chan, Haleem J. Isaaq, Timothy J. Waybright, Deborah Penque, Timothy D. Veenstra, and Tânia Simões
- Subjects
Proteomics ,Two-dimensional liquid chromatography ,Tandem mass spectrometry ,Biophysics ,Nasal epithelium ,Biology ,Cell Fractionation ,Biochemistry ,Article ,Cellular fractionation ,medicine ,Humans ,Chromatography ,Functional analysis ,Proteomic Profiling ,Gene Expression Profiling ,Membrane Proteins ,Upper and lower airways ,Epithelium ,Transmembrane protein ,Genómica Funcional e Estrutural ,Nasal Mucosa ,medicine.anatomical_structure ,Membrane protein ,Respiratory epithelium ,Cell fractionation ,Multidimensional chromatographic separations ,Chromatography, Liquid - Abstract
A comprehensive proteomic profiling of nasal epithelium (NE) is described. This study relies on simple subcellular fractionation used to obtain soluble- and membrane-enriched fractions followed by 2-dimensional liquid chromatography (2D-LC) separation and tandem mass spectrometry (MS/MS). The cells were collected using a brushing technique applied on NE of clinically evaluated volunteers. Subsequently, the soluble- and the membrane-protein enriched fractions were prepared and analyzed in parallel using 2D-LC-MS/MS. In a set of 1482 identified proteins, 947 (63.9%) proteins were found to be associated to membrane fraction. Grand average hydropathy value index (GRAVY) analysis, the transmembrane protein mapping and annotations of primary location deposited in the Human Protein Reference Database (HPRD) confirmed an enrichment of hydrophobic proteins on this dataset. Ingenuity Pathway Analysis (IPA) of soluble fraction revealed an enrichment of molecular and cellular functions associated with cell death, protein folding and drug metabolism while in membrane fraction showed an enrichment of functions associated with molecular transport, protein trafficking and cell-to-cell signaling and interaction. The IPA showed similar enrichment of functions associated with cellular growth and proliferation in both soluble and membrane subproteomes. This finding was in agreement with protein content analysis using exponentially modified protein abundance index (emPAI). A comparison of our data with previously published studies focusing on respiratory tract epithelium revealed similarities related to identification of proteins associated with physical barrier function and immunological defence. In summary, we extended the NE molecular profile by identifying and characterizing proteins associated to pivotal functions of a respiratory epithelium, including the control of fluid volume and ionic composition at the airways' surface, physical barrier maintenance, detoxification and immunological defence. The extent of similarities supports the applicability of a less invasive analysis of NE to assess prognosis and treatment response of lung diseases such as asthma, cystic fibrosis and chronic obstructive pulmonary disease., Graphical abstract This work represents the most complete proteome characterization of nasal epithelium (NE) describing its relevant functions. NE usefulness in lung biomedical research is highlighted based on observed similarities with bronchial epithelial proteome. Research highlights ► Sub-fractionation of nasal epithelial cells (NEC) to improve the detection of low abundance proteins. ► Global proteome characterization based on a gel-free SCX-RPLC- chromatography coupled with MS/MS approach. ► Characterization of cell membrane proteins using a methanol-based solubilization before tryptic digestion. ► Pathway Analysis to profile molecular and cellular functions overrepresented in nasal epithelium. ► Extensive comparison of our data with previously published studies focusing on respiratory tract epithelium.
- Published
- 2011
18. Bioconjugation and Detection of Lactosamine Moiety using α1,3-Galactosyltransferase Mutants That Transfer C2-Modified Galactose with a Chemical Handle
- Author
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Pradman K. Qasba, Marta Pasek, Timothy J. Waybright, Maria Manzoni, Boopathy Ramakrishnan, and Elizabeth Boeggeman
- Subjects
Models, Molecular ,Glycan ,Glycoconjugate ,Stereochemistry ,Molecular Sequence Data ,Mutant ,Biomedical Engineering ,Pharmaceutical Science ,Bioengineering ,Article ,chemistry.chemical_compound ,Glycolipid ,Glycosyltransferase ,Animals ,Humans ,Moiety ,Amino Acid Sequence ,Trisaccharide ,Pharmacology ,chemistry.chemical_classification ,Base Sequence ,biology ,Organic Chemistry ,Galactose ,Amino Sugars ,Galactosyltransferases ,carbohydrates (lipids) ,Kinetics ,chemistry ,Biochemistry ,Mutation ,biology.protein ,Cattle ,Sequence Alignment ,Biotechnology - Abstract
Studies on wild-type and mutant glycosyltransferases have shown that they can transfer modified sugars with a versatile chemical handle, such as keto or azido group, that can be used for conjugation chemistry and detection of glycan residues on glycoconjugates. To detect the most prevalent glycan epitope, N-acetyllactosamine (LacNAc (Galbeta1-4GalNAcbeta)), we have mutated a bovine alpha1,3-galactosyltransferse (alpha3Gal-T)() enzyme which normally transfers Gal from UDP-Gal to the LacNAc acceptor, to transfer GalNAc or C2-modified galactose from their UDP derivatives. The alpha3Gal-T enzyme belongs to the alpha3Gal/GalNAc-T family that includes human blood group A and B glycosyltransferases, which transfer GalNAc and Gal, respectively, to the Gal moiety of the trisaccharide Fucalpha1-2Galbeta1-4GlcNAc. On the basis of the sequence and structure comparison of these enzymes, we have carried out rational mutation studies on the sugar donor-binding residues in bovine alpha3Gal-T at positions 280 to 282. A mutation of His280 to Leu/Thr/Ser/Ala or Gly and Ala281 and Ala282 to Gly resulted in the GalNAc transferase activity by the mutant alpha3Gal-T enzymes to 5-19% of their original Gal-T activity. We show that the mutants (280)SGG(282) and (280)AGG(282) with the highest GalNAc-T activity can also transfer modified sugars such as 2-keto-galactose or GalNAz from their respective UDP-sugar derivatives to LacNAc moiety present at the nonreducing end of glycans of asialofetuin, thus enabling the detection of LacNAc moiety of glycoproteins and glycolipids by a chemiluminescence method.
- Published
- 2009
19. Chemical characterization of the aqueous algistatic fraction of barley straw (Hordeum vulgare) inhibiting Microcystis aeruginosa
- Author
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M. Drew Ferrier, Timothy J. Waybright, and Daniel E. Terlizzi
- Subjects
food and beverages ,Plant Science ,Fractionation ,Aquatic Science ,Biology ,Straw ,biology.organism_classification ,Hydrolysis ,Polyphenol ,Botany ,Bioassay ,Microcystis aeruginosa ,Phytotoxicity ,Food science ,Hordeum vulgare - Abstract
The algistatic properties of aqueous barley straw (Hordeum vulgare) extracts have been observed in laboratory studies and in situ. This reported algistatic property has been used by farmers and horticulturists to control algal blooms in various systems and has become standard practice in some areas. However, both inhibition and stimulation of algal growth in freshwater and marine species have been demonstrated. While the number of taxa known to be inhibited by barley straw has increased, comparatively little has been done to isolate and classify the compound(s) responsible for this algistatic effect. A microplate assay system using Microcystis aeruginosa was developed to isolate and identify the inhibitory components of barley straw extract. M. aeruginosa was selected for the bioassay because it is consistently inhibited by barley straw extract in studies conducted by our laboratory and others. The 24-well plate assay utilizes in vivo fluorescence monitoring with a TECAN GENios plate reader to determine chlorophyll-a levels in each culture. Fractionation and partial chemical characterization of inhibitory extracts suggests that the inhibitors are polyphenolics with molecular weights (MW) between 1,000 and 3,000 Da. Percolation of the aqueous extract through a Polyamide CC6 resin or through various MW cutoff filters resulted in the loss of algistatic activity, which confirms this assertion, while hydrolysis resulted in little change in the activity profile. Fractionation by HPLC methods yielded a highly potent multi-compound fraction, showing toxicity at 353 mg L−1 and algistatic activity between 11.1 and 3.53 mg L−1.
- Published
- 2008
20. Detection of Bladder Cancer in Human Urine by Metabolomic Profiling Using High Performance Liquid Chromatography/Mass Spectrometry
- Author
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Haleem J. Issaq, Alexander Kravstov, Brian T. Luke, Timothy J. Waybright, Timothy D. Veenstra, Ofer Nativ, Michael Mullerad, and Elias Issaq
- Subjects
Male ,medicine.medical_specialty ,Urology ,Urinary system ,Urine ,Mass spectrometry ,High-performance liquid chromatography ,Mass Spectrometry ,Metabolomics ,Humans ,Medicine ,Chromatography, High Pressure Liquid ,Aged ,Aged, 80 and over ,Urinary bladder ,Bladder cancer ,Chromatography ,business.industry ,Middle Aged ,medicine.disease ,Surgery ,medicine.anatomical_structure ,Transitional cell carcinoma ,Urinary Bladder Neoplasms ,Female ,business - Abstract
The current use of cystoscopy for screening and detecting bladder cancer is invasive and expansive. Various urine based biomarkers have been used for this purpose with limited success. Metabolomics, ie metabonomics, is the quantitative measurement of the metabolic response to pathophysiological stimuli. This analysis provides a metabolite pattern that can be characteristic of various benign and malignant conditions. We evaluated high performance liquid chromatography coupled online with a mass spectrometer metabolomic approach to differentiate urine samples from healthy individuals and patients with bladder cancer.Urine specimens were collected from 48 healthy individuals and 41 patients with transitional cell carcinoma, and stored at -80C. Samples were analyzed using an Agilent 1100 Series high performance liquid chromatography system (Agilent Technologies, Santa Clara, California) coupled online with a hybrid triple-quad time-of-flight QSTAR XL mass spectrometer. At the time of analysis samples were thawed and centrifuged. The resulting total ion chromatograms of each sample were submitted for statistical analysis. For data interpretation in this study 2 statistical methods were used, that is principal component analysis and orthogonal partial least square-discriminate analysis.Using positive ionization mass spectrometry orthogonal partial least square-discriminate analysis correctly predicted 48 of 48 healthy and 41 of 41 bladder cancer urine samples, while principal component analysis, which is an unsupervised profiling statistical method, confirmed these results and correctly predicted 46 of 48 healthy and 40 of 41 bladder cancer urine samples.The results of this proof of concept study in a relatively small number of subjects indicate that metabolomics using high performance liquid chromatography-mass spectrometry has the potential to become a noninvasive early detection test for bladder cancer.
- Published
- 2008
21. Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates
- Author
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Timothy D. Veenstra, Josip Blonder, Carol B. Fowler, Timothy J. O'Leary, Timothy J. Waybright, Robert E. Cunningham, and Jeffrey T. Mason
- Subjects
Tris ,Time Factors ,Hydrostatic pressure ,Tissue Banks ,Buffers ,Mass Spectrometry ,Pathology and Forensic Medicine ,Fixatives ,chemistry.chemical_compound ,Formaldehyde ,Aspartic acid ,Hydrostatic Pressure ,medicine ,Sodium dodecyl sulfate ,Molecular Biology ,chemistry.chemical_classification ,Paraffin Embedding ,Temperature ,Proteins ,Cell Biology ,Hydrogen-Ion Concentration ,Trypsin ,Amino acid ,chemistry ,Biochemistry ,Glycine ,Muramidase ,Lysozyme ,medicine.drug - Abstract
High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications to proteins. We previously reported a method for the formation of 'tissue surrogates' as a model to study formalin fixation, histochemical processing, and protein retrieval from FFPE tissues. In this study, we demonstrate the use of high hydrostatic pressure as a method for efficient protein recovery from FFPE tissue surrogates. Reversal of formaldehyde-induced protein adducts and crosslinks was observed when lysozyme tissue surrogates were extracted at 45 000 psi and 80-100 degrees C in Tris buffers containing 2% sodium dodecyl sulfate and 0.2 M glycine at pH 4. These conditions also produced peptides resulting from acid-catalyzed aspartic acid cleavage. Additives such as trimethylamine N-oxide or copper (II) chloride decreased the total percentage of these aspartic acid cleavage products, while maintaining efficient reversal of intermolecular crosslinks in the FFPE tissue surrogates. Mass spectrometry analysis of the recovered lysozyme yielded 70% sequence coverage, correctly identified all formaldehyde-reactive amino acids, and demonstrated hydrolysis at all of the expected trypsin cleavage sites. This study demonstrates that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues for proteomic analysis.
- Published
- 2008
22. Comparison of 1D and 2D NMR Spectroscopy for Metabolic Profiling
- Author
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Timothy J. Waybright, Que N. Van, Timothy D. Veenstra, Qiaoli Li, Michael Dean, Hong Lou, Qiujie Jiang, Gary M. Muschik, Haleem J. Issaq, and Jouni Uitto
- Subjects
Mice, Knockout ,Magnetic Resonance Spectroscopy ,Proteome ,Metabolite ,Large dynamic range ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Biochemistry ,Disease Models, Animal ,Mice ,chemistry.chemical_compound ,Metabolomics ,Nuclear magnetic resonance ,chemistry ,Proton NMR ,Animals ,ATP-Binding Cassette Transporters ,Multidrug Resistance-Associated Proteins ,Protons ,Pseudoxanthoma Elasticum ,Spectroscopy ,Two-dimensional nuclear magnetic resonance spectroscopy ,Macromolecule - Abstract
High-resolution, liquid state nuclear magnetic resonance (NMR) spectroscopy is a popular platform for metabolic profiling because the technique is nondestructive, quantitative, reproducible, and the spectra contain a wealth of biochemical information. Because of the large dynamic range of metabolite concentrations in biofluids, statistical analyses of one-dimensional (1D) proton NMR data tend to be biased toward selecting changes in more abundant metabolites. Although two-dimensional (2D) proton-proton experiments can alleviate spectral crowding, they have been mainly used for structural determination. In this study, 2D total correlation spectroscopy NMR was used to compare the global metabolic profiles of urine obtained from wild-type and Abcc6-knockout mice. The 2D data were compared to an improved 1D experiment in which signal contributions from macromolecules and the urea peak have been spectroscopically removed for more accurate quantitation of low-abundance metabolites. Although statistical models from both 1D and 2D data could differentiate samples acquired from the two groups of mice, only the 2D spectra allowed the characterization of statistically relevant changes in the low-abundance metabolites. While acquisition of the 2D data require more time, the data obtained resulted in a more meaningful and comprehensive metabolic profile, aided in metabolite identifications, and minimized ambiguities in peak assignments.
- Published
- 2007
23. LC‐MS in Metabonomics: Optimization of Experimental Conditions for the Analysis of Metabolites in Human Urine
- Author
-
Timothy J. Waybright, Que N. Van, Thomas P. Conrads, Gary M. Muschik, Timothy D. Veenstra, and Haleem J. Issaq
- Subjects
Chromatography ,Chemistry ,Metabolite ,Clinical Biochemistry ,Pharmaceutical Science ,Reversed-phase chromatography ,Mass spectrometry ,Biochemistry ,High-performance liquid chromatography ,Biological fluid ,Analytical Chemistry ,chemistry.chemical_compound ,Metabolomics ,Liquid chromatography–mass spectrometry - Abstract
The analysis of metabolic pathways for dysfunction has been used for many years in the scientific and medical community to determine overall health. Metabonomics (metabolomics), the global profiling of metabolites, has experienced a rekindling of interest due, in part, to advances in analytical instrumentation for conducting measurements and informatics available for interpretation of the data acquired in this area of biomedical research. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) based approaches are two primary analytical methods of choice for conducting metabonomic measurements. To overcome the complexity and wide dynamic range of concentrations of metabolites present in biological samples, a common practice is to couple online an analytical separation, typically high performance liquid chromatography (HPLC), with the mass spectrometer. Hence, of critical importance are not only the MS acquisition parameters, but also optimization of those variables that impact th...
- Published
- 2006
24. Combined Chemical and Enzymatic Stable Isotope Labeling for Quantitative Profiling of Detergent-Insoluble Membrane Proteins Isolated Using Triton X-100 and Brij-96
- Author
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David A. Lucas, Frances J. Sharom, Josip Blonder, King C. Chan, Haleem J. Issaq, Galina Radeva, Li-Rong Yu, Timothy J. Waybright, and Timothy D. Veenstra
- Subjects
Proteomics ,Spectrometry, Mass, Electrospray Ionization ,Octoxynol ,Electrospray ionization ,Detergents ,Molecular Sequence Data ,Quantitative proteomics ,Biotin ,Biochemistry ,Chromatography, Affinity ,Article ,Polyethylene Glycols ,chemistry.chemical_compound ,Membrane Microdomains ,Oxygen Radioisotopes ,Cell Line, Tumor ,medicine ,Animals ,Plant Oils ,Trypsin ,Amino Acid Sequence ,Carbon Radioisotopes ,Peptide sequence ,Chromatography ,Chemistry ,Membrane Proteins ,General Chemistry ,Deuterium ,Rats ,Membrane protein ,Isotope Labeling ,Proteome ,Triton X-100 ,medicine.drug - Abstract
Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five non-cysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.
- Published
- 2006
25. Abstract 1366: Structural basis of impaired GTP hydrolysis in oncogenic mutants of KRAS
- Author
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Oleg Chertov, Dhirendra K. Simanshu, Timothy H. Tran, Timothy J. Waybright, Andrew G. Stephen, William K. Gillette, Dwight V. Nissley, Dominic Esposito, Frank McCormick, and Sathiya Dharmaiah
- Subjects
Cancer Research ,Oncology ,Biochemistry ,Chemistry ,Mutant ,medicine ,GTPase ,KRAS ,medicine.disease_cause - Abstract
RAS mutations are found in one-third of all human cancers. Among the three RAS isoforms - HRAS, KRAS and NRAS, KRAS is the most commonly mutated gene (in 86% of RAS-driven cancers) and the mutations are often detected in pancreatic, colorectal and lung cancers. RAS proteins function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. The active or inactive state of RAS proteins is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In the GTP-bound state, RAS proteins interact with a variety of effector proteins, such as Raf, PI3K, and RalGDS, leading to activation of several signaling cascades within the cell. In 98% of the cases, oncogenic RAS mutations are found at amino acid positions G12, G13, and Q61 which impair intrinsic and GAP-mediated GTPase function resulting in accumulation of constitutively GTP-bound RAS in cells. To gain insights into the effect of oncogenic mutations on overall structure and GTP hydrolysis, we solved high-resolution crystal structures of wild type and six oncogenic mutants (G12C, G12D, G12V, G13D, Q61L and Q61R) of KRAS4b in complex with GMPPNP (a non-hydrolysable GTP analog) and magnesium. Comparison of GDP and GMPPNP bound structures of wild type (WT) KRAS4b suggests conformational changes that occur when KRAS4b transitions from inactive to active state. Comparison of mutant and wild-type KRAS structures has shown the appearance of new pockets in some cases that could be exploited for structure-based drug design. Structural superposition of mutants vs. wild type KRAS4b in complex with GMPPNP/Mg2+, and KRAS4b mutants vs. WT-HRAS bound to RASA1-GAP provides a rationale for impaired intrinsic and GAP-mediated GTP hydrolysis in the KRAS mutants. Citation Format: Timothy Tran, Sathiya Dharmaiah, Oleg Chertov, Timothy Waybright, William Gillette, Dominic Esposito, Dwight Nissley, Frank McCormick, Andrew Stephen, Dhirendra Simanshu. Structural basis of impaired GTP hydrolysis in oncogenic mutants of KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1366. doi:10.1158/1538-7445.AM2017-1366
- Published
- 2017
26. Preparation of the low molecular weight serum proteome for mass spectrometry analysis
- Author
-
Timothy J, Waybright, King C, Chan, Timothy D, Veenstra, and Zhen, Xiao
- Subjects
Molecular Weight ,Proteomics ,Proteome ,Humans ,Biomarkers ,Mass Spectrometry - Abstract
The discovery of viable biomarkers or indicators of disease states is complicated by the inherent complexity of the chosen biological specimen. Every sample, whether it is serum, plasma, urine, tissue, cells, or a host of others, contains thousands of large and small components, each interacting in multiple ways. The need to concentrate on a group of these components to narrow the focus on a potential biomarker candidate becomes, out of necessity, a priority, especially in the search for immune-related low molecular weight serum biomarkers. One such method in the field of proteomics is to divide the sample proteome into groups based on the size of the protein, analyze each group, and mine the data for statistically significant items. This chapter details a portion of this method, concentrating on a method for fractionating and analyzing the low molecular weight proteome of human serum.
- Published
- 2013
27. Preparation of human cerebrospinal fluid for proteomics biomarker analysis
- Author
-
Timothy J, Waybright
- Subjects
Proteomics ,Chromatography, Reverse-Phase ,Proteome ,Alzheimer Disease ,Amyotrophic Lateral Sclerosis ,Humans ,Cerebrospinal Fluid Proteins ,Biomarkers ,Mass Spectrometry ,Specimen Handling - Abstract
The analysis of the cerebrospinal fluid (CSF) proteome in recent years has resulted in a valuable repository of data for targeting and diagnosing a variety of diseases, such as Parkinson's disease, Alzheimer's disease, traumatic brain injury, and amyotrophic lateral sclerosis. Human ventricular CSF contains numerous proteins that are unique to CSF due in part to the interaction of the biofluid with the brain. This allows researchers to obtain information from a region that would otherwise be inaccessible except through invasive surgery or during autopsy. Characterization of the CSF proteome requires that strict care be taken so that sample integrity and fidelity are maintained to ensure data reproducibility. Standardized methods in sample collection, storage, preparation, analysis, and data mining must be used for meaningful information to be obtained. The following method describes a simple and robust approach for preparing CSF samples for analysis via reversed-phase liquid chromatography (RPLC) and mass spectrometry (MS).
- Published
- 2013
28. Preparation of human serum for prolactin measurement by multiple reaction monitoring mass spectrometry
- Author
-
Timothy J, Waybright, Xia, Xu, Jessica M, Faupel-Badger, and Zhen, Xiao
- Subjects
Humans ,Protein Isoforms ,Mass Spectrometry ,Peptide Fragments ,Chromatography, Liquid ,Prolactin - Abstract
The measurement of the protein hormone prolactin (PRL) in biological samples has developed over the years into a routine clinical assay aiding the diagnosis of multiple medical conditions. PRL is known to exist in multiple isoforms circulating throughout the body. Current methodologies for measuring the PRL levels typically involve a variety of immunoassays. However, most of these tests are not capable of distinguishing between the different isoforms. To address this need, we have developed a highly specialized method employing multiple reaction monitoring mass spectrometry (MRM-MS) capable of monitoring seven distinct peptides from two of the most common prolactin isoforms (the 23 kDa PRL and its 16 kDa N-terminal cleavage product). Since serum is the main source of clinical specimen for the measurement of prolactin isoforms, the method described in this chapter is focused on the approach to processing whole serum samples for prolactin analysis via reversed-phase liquid chromatography (RPLC) and MRM-MS.
- Published
- 2013
29. Preparation of Human Cerebrospinal Fluid for Proteomics Biomarker Analysis
- Author
-
Timothy J. Waybright
- Subjects
Cerebrospinal fluid ,Traumatic brain injury ,business.industry ,Proteome ,medicine ,Sample collection ,Biomarker Analysis ,Amyotrophic lateral sclerosis ,Alzheimer's disease ,medicine.disease ,business ,Proteomics ,Bioinformatics - Abstract
The analysis of the cerebrospinal fluid (CSF) proteome in recent years has resulted in a valuable repository of data for targeting and diagnosing a variety of diseases, such as Parkinson's disease, Alzheimer's disease, traumatic brain injury, and amyotrophic lateral sclerosis. Human ventricular CSF contains numerous proteins that are unique to CSF due in part to the interaction of the biofluid with the brain. This allows researchers to obtain information from a region that would otherwise be inaccessible except through invasive surgery or during autopsy. Characterization of the CSF proteome requires that strict care be taken so that sample integrity and fidelity are maintained to ensure data reproducibility. Standardized methods in sample collection, storage, preparation, analysis, and data mining must be used for meaningful information to be obtained. The following method describes a simple and robust approach for preparing CSF samples for analysis via reversed-phase liquid chromatography (RPLC) and mass spectrometry (MS).
- Published
- 2013
30. Preparation of the Low Molecular Weight Serum Proteome for Mass Spectrometry Analysis
- Author
-
Timothy J. Waybright, King C. Chan, Timothy D. Veenstra, and Zhen Xiao
- Subjects
Chemistry ,Serum proteome ,Sample (material) ,Proteome ,Computational biology ,Visual symptoms ,Mass spectrometry ,Protein depletion - Abstract
The ability to cure or manage many diseases is highly dependent on the ability to correctly diagnose them at the earliest possible stage. Diagnosis relies heavily on biomarkers whether these be visual symptoms or molecules found within samples acquired from the patient. For conditions that lack useful biomarkers, researchers are often faced with the task of sifting through very complex biological samples (i.e., serum, plasma, urine, tissue, cells, etc.) with the hope of discovering a small number of molecules that are exquisitely diagnostic for the condition of interest. One discovery strategy that has been frequently used is to fractionate the biological samples being studied into simpler aliquots that can be more easily characterized using existing technologies. One such fractionation method is to isolate a specific portion based on a specific property (i.e., size, phosphorylation state, charge, etc.) of the proteins within the sample. This method provides a simplified sample that can be characterized at a higher coverage level than the complex sample from which it was derived. This chapter details one of these methods, the extraction and analysis of the low molecular weight proteome of human serum.
- Published
- 2013
31. Preparation of Human Serum for Prolactin Measurement by Multiple Reaction Monitoring Mass Spectrometry
- Author
-
Zhen Xiao, Timothy J. Waybright, Xia Xu, and Jessica M. Faupel-Badger
- Subjects
Gene isoform ,endocrine system ,Biochemistry ,Chemistry ,Prolactin blood ,Selected reaction monitoring ,Mass spectrometry ,Serum samples ,hormones, hormone substitutes, and hormone antagonists ,Prolactin ,Prolactin measurement ,Hormone - Abstract
The measurement of the protein hormone prolactin (PRL) in biological samples has developed over the years into a routine clinical assay aiding the diagnosis of multiple medical conditions. PRL is known to exist in multiple isoforms circulating throughout the body. Current methodologies for measuring the PRL levels typically involve a variety of immunoassays. However, most of these tests are not capable of distinguishing between the different isoforms. To address this need, we have developed a highly specialized method employing multiple reaction monitoring mass spectrometry (MRM-MS) capable of monitoring seven distinct peptides from two of the most common prolactin isoforms (the 23 kDa PRL and its 16 kDa N-terminal cleavage product). Since serum is the main source of clinical specimen for the measurement of prolactin isoforms, the method described in this chapter is focused on the approach to processing whole serum samples for prolactin analysis via reversed-phase liquid chromatography (RPLC) and MRM-MS.
- Published
- 2013
32. Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis
- Author
-
Timothy J. Waybright, Timothy D. Veenstra, Carol B. Fowler, Timothy J. O'Leary, and Jeffrey T. Mason
- Subjects
Proteomics ,high-pressure protein extraction ,Hot Temperature ,Formalin fixed paraffin embedded ,Proteome ,Hydrostatic pressure ,Biology ,FFPE ,Biochemistry ,03 medical and health sciences ,Mice ,0302 clinical medicine ,mouse liver ,Formaldehyde ,Protein purification ,Hydrostatic Pressure ,Technical Note ,Animals ,030304 developmental biology ,mass spectrometry ,0303 health sciences ,Paraffin Embedding ,Extraction (chemistry) ,Disease progression ,Proteins ,General Chemistry ,formalin-fixed paraffin-embedded ,Molecular biology ,Liver ,030220 oncology & carcinogenesis ,Archival tissue ,heat and pressure - Abstract
Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.
- Published
- 2012
33. Cancer biomarker discovery: Opportunities and pitfalls in analytical methods
- Author
-
Timothy J. Waybright, Haleem J. Issaq, and Timothy D. Veenstra
- Subjects
Electrophoresis ,Proteomics ,Clinical tests ,Clinical Biochemistry ,Disease progression ,Cancer ,Biology ,Bioinformatics ,medicine.disease ,Biochemistry ,Mass Spectrometry ,Analytical Chemistry ,Clinical trial ,Mice ,Metabolomics ,Neoplasms ,medicine ,Biomarkers, Tumor ,Biomarker (medicine) ,Animals ,Humans ,Biomarker discovery ,Chromatography, High Pressure Liquid - Abstract
Many diseases result in specific and characteristic changes in the chemical and biochemical profiles of biological fluids and tissues prior to development of clinical symptoms. These changes are often useful diagnostic and prognostic biomarkers. Identifying biomarkers that can be used for the early detection of cancer will result in more efficient treatments, reduction in suffering, and lower mortality rates. An ideal screening test should be non-invasive with high sensitivity and specificity. Proteomic and metabolomic analyses of biological samples can reveal changes in abundance levels of metabolites and proteins that when validated and confirmed through clinical trials can function as clinical tests for early detection, diagnosis, monitoring disease progression, and predicting therapeutic response. While the past decade has seen great advancements in proteomics and metabolomics research producing potential biomarkers for cancer, most of the identified biomarkers have failed to replace existing clinical tests. To become a clinically approved test, a potential biomarker should be confirmed and validated using hundreds of specimens and should be reproducible, specific, and sensitive. A search of the scientific and medical literature indicates that many studies report the discovery of potential biomarkers without proper validation and/or they do not meet the above criteria. In this manuscript, we will discuss the successes and the pitfalls of biomarker research and comment on study and experimental design, which in most cases is lacking, resulting in suboptimal biomarkers.
- Published
- 2010
34. Characterization of the human ventricular cerebrospinal fluid proteome obtained from hydrocephalic patients
- Author
-
Timothy J. Waybright, Richard G. Ellenbogen, Richard S. Morrison, Bradley J. Hollinger, Timothy D. Veenstra, and Anthony M. Avellino
- Subjects
Proteomics ,Pathology ,medicine.medical_specialty ,Proteome ,Traumatic brain injury ,Biophysics ,Biochemistry ,Mass Spectrometry ,Cerebrospinal fluid ,Cations ,Medicine ,Humans ,Amyotrophic lateral sclerosis ,Child ,business.industry ,Infant, Newborn ,Infant ,Cerebrospinal Fluid Proteins ,medicine.disease ,Spinal cord ,Chromatography, Ion Exchange ,Lipids ,Hydrocephalus ,Traumatic injury ,medicine.anatomical_structure ,Child, Preschool ,business ,Algorithms - Abstract
The continuing expansion of proteomic technology has been fueled by the potential for discovering novel biomarkers that may be used for the early detection of disease. It has been proposed that human cerebrospinal fluid (CSF), which surrounds and protects the brain and spinal cord from traumatic injury, may be a valuable target for the diagnosis of a variety of conditions such as Alzheimer's disease, traumatic brain injury, amyotrophic lateral sclerosis and Parkinson's disease. The immense complexity of biofluids, however, still requires that considerable development be made in the analytical techniques used so that comprehensive coverage of the proteins present in such samples is achieved. Using a simple separation strategy the protein complement of human ventricular cerebrospinal fluid obtained from patients with hydrocephalus was evaluated. The study resulted in the identification of over 1500 unique proteins that were found within all nine CSF samples that were analyzed. Comparison with the HUPO serum proteome database demonstrated that human ventricular CSF contains a large array of proteins that may be unique to CSF. This analysis greatly increases our knowledge of the protein content of this clinically important biofluid.
- Published
- 2009
35. Analytical and statistical approaches to metabolomics research
- Author
-
Timothy J. Waybright, Que N. Van, Gary M. Muschik, Timothy D. Veenstra, and Haleem J. Issaq
- Subjects
Chromatography ,Biomedical Research ,Chemistry ,Data interpretation ,Electrophoresis, Capillary ,Filtration and Separation ,Computational biology ,Metabolite analysis ,Gas Chromatography-Mass Spectrometry ,Analytical Chemistry ,Specimen Handling ,Chemometrics ,Metabolomics ,Principal component analysis ,Profiling (information science) ,Humans ,Sample collection ,Two sample ,Nuclear Magnetic Resonance, Biomolecular ,Biomarkers ,Chromatography, High Pressure Liquid ,Software - Abstract
Metabolomics, the global profiling of metabolites in different living systems, has experienced a rekindling of interest partially due to the improved detection capabilities of the instrumental techniques currently being used in this area of biomedical research. The analytical methods of choice for the analysis of metabolites in search of disease biomarkers in biological specimens, and for the study of various low molecular weight metabolic pathways include NMR spectroscopy, GC/MS, CE/MS, and HPLC/MS. Global metabolite analysis and profiling of two different sets of data results in a plethora of data that is difficult to manage or interpret manually because of their subtle differences. Multivariate statistical methods and pattern-recognition programs were developed to handle the acquired data and to search for the discriminating features between data acquired from two sample sets, healthy and diseased. Metabolomics have been used in toxicology, plant physiology, and biomedical research. In this paper, we discuss various aspects of metabolomic research including sample collection, handling, storage, requirements for sample analysis, peak alignment, data interpretation using statistical approaches, metabolite identification, and finally recommendations for successful analysis.
- Published
- 2009
36. Overcoming problems of compound storage in DMSO: solvent and process alternatives
- Author
-
John R. Britt, Timothy J. Waybright, and Thomas G. McCloud
- Subjects
Molar concentration ,Drug Storage ,Biochemistry ,Mass Spectrometry ,Analytical Chemistry ,Absorption ,Viscosity ,Cell Line, Tumor ,Bioassay ,Humans ,Dimethyl Sulfoxide ,Solubility ,Process engineering ,Cell Proliferation ,Chromatography ,Precipitation (chemistry) ,Chemistry ,business.industry ,Temperature ,Water ,Reference Standards ,Solvent ,Solutions ,Pharmaceutical Preparations ,Compatibility (mechanics) ,Solvents ,Molecular Medicine ,business ,Biotechnology - Abstract
The common practice of preparing storage libraries of compounds in 100% DMSO solution well in advance of bioassay brings with it difficulties that affect the accuracy of the data obtained. This publication presents a series of studies done on a subset of compounds that are difficult to bioassay because they precipitate from DMSO solution. These compounds are members of a frequently used, diverse compound library of the sort commonly used in the high-throughput screening (HTS) environment. Experiments were performed to determine the concentration of drug in solution above the precipitate, observe the time course and effect of various mixtures of solvents upon precipitation, measure the viscosity of cosolvents to determine compatibility with HTS, determine water absorption rates for various solvent combinations, and investigate resolubilization techniques to ensure proper drug solution for HTS. Recommendations are made on how to best maximize the probability that problem compounds will remain in solution, be accurately transferred during assay plate production, and, as a result, be accurately bioassayed at the specified molar concentration.
- Published
- 2009
37. Bringing protein biomarker discovery to the clinic
- Author
-
Timothy J. Waybright and Timothy D. Veenstra
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Electrospray ionization ,education ,Computational biology ,Biology ,Bioinformatics ,Mass spectrometry ,Sensitivity and Specificity ,Mass Spectrometry ,Pathology and Forensic Medicine ,Kidney Tubules, Proximal ,Genetics ,Humans ,Amino Acid Sequence ,Biomarker discovery ,Molecular Biology ,Peptide sequence ,Gene ,Carcinoma, Renal Cell ,health care economics and organizations ,Proteins ,humanities ,Kidney Neoplasms ,ComputingMethodologies_PATTERNRECOGNITION ,Molecular Medicine ,Biomarkers - Abstract
This era is a very exciting time to be a life scientist. This opinion is primarily based on the technologies that enable information on hundreds of proteins (or genes, transcripts and metabolites) ...
- Published
- 2009
38. Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection
- Author
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Pradman K. Qasba, Elizabeth Boeggeman, Anu Puri, Timothy J. Waybright, Kristin H. Loomis, Marta Pasek, Boopathy Ramakrishnan, and Maria Manzoni
- Subjects
PNGase F ,Vascular Endothelial Growth Factor A ,Glycan ,Glycosylation ,medicine.drug_class ,Receptor, ErbB-2 ,Population ,Biomedical Engineering ,Pharmaceutical Science ,Oligosaccharides ,Bioengineering ,Enzyme-Linked Immunosorbent Assay ,Monoclonal antibody ,Article ,Substrate Specificity ,chemistry.chemical_compound ,Polysaccharides ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Biotinylation ,education ,Fluorescent Dyes ,Pharmacology ,education.field_of_study ,Glucosamine ,Binding Sites ,biology ,Staining and Labeling ,Organic Chemistry ,Antibodies, Monoclonal ,Galactose ,Glycosyltransferases ,Molecular biology ,Sialic acid ,chemistry ,Biochemistry ,Immunoglobulin G ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Antigens, Surface ,biology.protein ,Mutant Proteins ,Biotechnology - Abstract
The Fc N-glycan chains of four therapeutic monoclonal antibodies (mAbs), namely, Avastin, Rituxan, Remicade, and Herceptin, released by PNGase F, show by MALDI analysis that these biantennary N-glycans are a mixture of G0, G1, and G2 glycoforms. The G0 glycoform has no galactose on the terminal GlcNAc residues, and the G1 and G2 glycoforms have one or two terminal galactose residues, respectively, while no N-glycan with terminal sialic acid residue is observed. We show here that under native conditions we can convert the N-glycans of these mAbs to a homogeneous population of G0 glycoform using beta1,4 galactosidase from Streptococcus pneumoniae. The G0 glycoforms of mAbs can be galactosylated with a modified galactose having a chemical handle at the C2 position, such as ketone or azide, using a mutant beta1,4-galactosyltransferase (beta1,4Gal-T1-Y289L). The addition of the modified galactose at a specific glycan residue of a mAb permits the coupling of a biomolecule that carries an orthogonal reactive group. The linking of a biotinylated or a fluorescent dye carrying derivatives selectively occurs with the modified galactose, C2-keto-Gal, at the heavy chain of these mAbs, without altering their antigen binding activities, as shown by indirect enzyme linked immunosorbent assay (ELISA) and fluorescence activated cell sorting (FACS) methods. Our results demonstrate that the linking of cargo molecules to mAbs via glycans could prove to be an invaluable tool for potential drug targeting by immunotherapeutic methods.
- Published
- 2009
39. Identification of highly expressed, soluble proteins using an improved, high-throughput pooled ORF expression technology
- Author
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Robert M. Stephens, Dominic Esposito, Timothy J. Waybright, Timothy D. Veenstra, David A. Lucas, James L. Hartley, and William K. Gillette
- Subjects
Proteomics ,Biology ,medicine.disease_cause ,Tandem mass spectrometry ,Transfection ,General Biochemistry, Genetics and Molecular Biology ,Open Reading Frames ,Tandem Mass Spectrometry ,Gene expression ,medicine ,Escherichia coli ,Humans ,Trypsin ,ORFS ,Cloning, Molecular ,Chromatography, High Pressure Liquid ,Cloning ,Recombination, Genetic ,Proteins ,Reproducibility of Results ,Molecular biology ,Open reading frame ,Solubility ,Peptides ,Biotechnology ,medicine.drug - Abstract
This article describes an improved pooled open reading frame (ORF) expression technology (POET) that uses recombinational cloning and solution-based tandem mass spectrometry (MS/MS) to identify ORFs that yield high levels of soluble, purified protein when expressed in Escherichia coli. Using this method, three identical pools of 512 human ORFs were subcloned, purified, and transfected into three separate E. coli cultures. After bulk expression and purification, the proteins from the three separate pools were digested into tryptic peptides. Each of these samples was subsequently analyzed in triplicate using reversed-phase high-performance liquid chromatography (LC) coupled directly online with MS/MS. The abundance of each protein was determined by calculating the average exponentially modified protein abundance index (emPAI) of each protein across the three protein pools. Human proteins that consistently gave high emPAI values were subjected to small-scale expression and purification. These clones showed high levels of expression of soluble protein. Conversely, proteins that were not observed by LC-MS/MS did not show any detectable soluble expression in small-scale validation studies. Using this improved POET method allows the expression characteristics of hundreds of proteins to be quickly determined in a single experiment.
- Published
- 2008
40. 888: Metabolomic Urine Samples Profiling Using High Performance Liquid Chromatography Coupled with Mass Spectrometry to Detect Bladder Cancer
- Author
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Michael Mullerad, Timothy J. Waybright, Brian Luke, Ofer Nativ, Alexander Kravtsov, Timothy D. Veenstra, Haleem J. Issaq, and Elias Issaq
- Subjects
Chromatography ,Metabolomics ,Bladder cancer ,business.industry ,Urology ,Medicine ,Urine ,business ,Mass spectrometry ,medicine.disease ,High-performance liquid chromatography - Published
- 2007
41. Site Specific Conjugation of Fluoroprobes to the Remodeled Fc N-Glycans of Monoclonal Antibodies Using Mutant Glycosyltransferases: Application for Cell Surface Antigen Detection.
- Author
-
Elizabeth Boeggeman, Boopathy Ramakrishnan, Marta Pasek, Maria Manzoni, Anu Puri, Kristin H. Loomis, Timothy J. Waybright, and Pradman K. Qasba
- Published
- 2009
- Full Text
- View/download PDF
42. Comparison of 1D and 2D NMR Spectroscopy for Metabolic Profiling.
- Author
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Que N. Van, Haleem J. Issaq, Qiujie Jiang, Qiaoli Li, Gary M. Muschik, Timothy J. Waybright, Hong Lou, Michael Dean, Jouni Uitto, and Timothy D. Veenstra
- Published
- 2008
- Full Text
- View/download PDF
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