28 results on '"Fonslow, Bryan R."'
Search Results
2. Capillary electrophoresis–mass spectrometry for direct structural identification of serum N-glycans
- Author
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Snyder, Christa M., Zhou, Xiaomei, Karty, Jonathan A., Fonslow, Bryan R., Novotny, Milos V., and Jacobson, Stephen C.
- Published
- 2017
- Full Text
- View/download PDF
3. Versatile strategy for controlling the specificity and activity of engineered T cells
- Author
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Ma, Jennifer S. Y., Kim, Ji Young, Kazane, Stephanie A., Choi, Sei-hyun, Yun, Hwa Young, Kim, Min Soo, Rodgers, David T., Pugh, Holly M., Singer, Oded, Sun, Sophie B., Fonslow, Bryan R., Kochenderfer, James N., Wright, Timothy M., Schultz, Peter G., Young, Travis S., Kim, Chan Hyuk, and Cao, Yu
- Published
- 2016
4. DAF-16/FOXO and HLH-30/TFEB function as combinatorial transcription factors to promote stress resistance and longevity
- Author
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Lin, Xin-Xuan, Sen, Ilke, Janssens, Georges E., Zhou, Xin, Fonslow, Bryan R., Edgar, Daniel, Stroustrup, Nicholas, Swoboda, Peter, Yates, 3rd, John R., Ruvkun, Gary, and Riedel, Christian G.
- Published
- 2018
- Full Text
- View/download PDF
5. The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity
- Author
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Heimbucher, Thomas, Liu, Zheng, Bossard, Carine, McCloskey, Richard, Carrano, Andrea C., Riedel, Christian G., Tanasa, Bogdan, Klammt, Christian, Fonslow, Bryan R., Riera, Celine E., Lillemeier, Bjorn F., Kemphues, Kenneth, Yates, John R., III, O’Shea, Clodagh, Hunter, Tony, and Dillin, Andrew
- Published
- 2015
- Full Text
- View/download PDF
6. Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes
- Author
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Fonslow, Bryan R., Kang, Seong A., Gestaut, Daniel R., Graczyk, Beth, Davis, Trisha N., Sabatini, David M., and Yates, John R., III
- Subjects
Electrochemistry -- Research ,Separation (Technology) -- Methods ,Separation (Technology) -- Technology application ,Proteins -- Chemical properties ,Coordination compounds -- Chemical properties ,Technology application ,Chemistry - Abstract
Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tsg and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15[degrees]C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection ( 10.1021/ac101235k
- Published
- 2010
7. Measuring aptamer equilibria using gradient micro free flow electrophoresis
- Author
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Turgeon, Ryan T., Fonslow, Bryan R., Jing, Meng, and Bowser, Michael T.
- Subjects
Chemistry - Abstract
Gradient micro free flow electrophoresis ([mu]FFE) was used to observe the equilibria of DNA aptamera with their targets (IgE or HIVRT) across a range of ligand concentrations. A continuous stream of aptamer was mixed online with an increasing concentration of target and introduced into the [mu]FFE device, which separated ligand--aptamer complexes from the unbound aptamer. The continuous nature of [mu]FFE allowed the equilibrium distribution of aptamer and complex to be measured at 300 discrete target concentrations within 5 min. This is a significant improvement in speed and precision over affinity capillary electrophoresis (ACE) assays. The dissociation constant of the aptamer--IgE complex was estimated to be 48 [+ or -] 3 nM. The high coverage across the range of ligand concentrations allowed complex stoichiometries of the aptamer--HIVRT complexes to be observed. Nearly continuous observation of the equilibrium distribution from 0 to 500 nM HIVRT revealed the presence of complexes with 3:1 (aptamer/HIVRT), 2:1, and 1:1 stoichiometries. 10.1021/ac902877v
- Published
- 2010
8. Fast determination of mitochondria electrophoretic mobility using micro free-flow electrophoresis
- Author
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Kostal, Vratislav, Fonslow, Bryan R., Arriaga, Edgar A., and Bowser, Michael T.
- Subjects
Electrophoresis -- Methods ,Mitochondria -- Properties ,Electrochemical analysis -- Methods ,Electron mobility -- Observations ,Chemistry - Abstract
Fast, continuous separation of mitochondria from rat myoblasts using micro free-flow electrophoresis ([micro]FFE) with online laser-induced fluorescence detection (LIF) is reported. Mitochondrial electrophoretic profiles were acquired in less than 30 s. In comparison to macroscale FFE instruments, [micro]FFE devices consumed approximately l00-fold less sample, used l0-fold less buffer, and required a 15-fold lower electric field. Mitochondrial electrophoretic mobility distributions measured using [micro]FFE were compared to those measured with a capillary electrophoresis instrument with laser-induced fluorescence detection (CE-LIF). There was high similarity between the two distributions with CE-LIF distribution being offset by 1.8 x [10.sup.-4] [cm.sup.2] [V.sup.-1] [s.sup.-1] with respect to the [micro]FFE distribution. We hypothesize that this offset results from the differences in electric field strength used in the techniques. In comparison to CE-LIF, analysis of mitochondria using [micro]FFE greatly decreased separation time and required less separation voltage, while maintaining low sample (125 nL) and buffer (250 [micro]L) volumes. These features together with the potential for collecting separated organelle fractions for further characterization make [micro]FFE a very attractive tool for the high-throughput analysis of organelle subpopulations as well as investigating the fundamentals of the electrophoretic mobility of biological particles. 10.1021/ac901508x
- Published
- 2009
9. Fast electrophoretic separation optimization using gradient micro free-flow electrophoresis
- Author
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Fonslow, Bryan R. and Bowser, Michael T.
- Subjects
Electrophoresis -- Methods ,Chemistry - Abstract
The continuous nature of micro free-flow electrophoresis ([micro]-FFE) was used to monitor the effect of a gradient of buffer conditions on the separation. This unique application has great potential for fast optimization of separation conditions and estimation of equilibrium constants. COMSOL was used to model pressure profiles in the development of a new [micro]-FFE design that allowed even application of a buffer gradient across the separation channel. The new design was fabricated in an all glass device using our previously published multiple-depth etch method (Fonslow, B. R.; Barocas, V. H.; Bowser, M. T. Anal. Chem. 2006, 78, 5369-5374, ref 1). Fluorescein solutions were used to characterize the applied gradients in the separation channel. Linear gradients were observed when buffer conditions were varied over a period of 5-10 min. The effect of a gradient of 0-50 mM hydroxypropyl-[beta]-cyclodextrin (HP-[beta]-CD) on the separation of a group of 4-fluoro7-nitro-2,1,3-benzoxadiazole (NBD-F) labeled primary amines was monitored as a proof of concept experiment. Direct comparisons to capillary electrophoresis (CE) separations performed under the same conditions were made. Gradient [micro]-FFE recorded 60 separations during a 5 min gradient allowing nearly complete coverage across a range of HP-[beta]-CD concentrations. In comparison, 4 h were required to assess 15 sets of conditions across the same range of HP-[beta]-CD concentrations using CE. Qualitatively, [micro]-FFE separations were predictive of the migration order and spacing of peaks in CE electropherograms measured under the same conditions. Data were fit to equations describing 1:1 analyte-additive binding to allow a more quantitative comparison between gradient [micro]-FFE and CE.
- Published
- 2008
10. Optimizing band width and resolution in micro-free flow electrophoresis
- Author
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Fonslow, Bryan R. and Bowser, Michael T.
- Subjects
Electrophoresis -- Usage ,Hydrodynamics -- Analysis ,Bandwidth -- Measurement ,Hydrofoil boats -- Hydrodynamics ,Hydrofoil boats -- Analysis ,Bandwidth allocation ,Bandwidth technology ,Chemistry - Abstract
The broadening mechanisms for micro-free flow electrophoresis ([micro]-FFE) have been investigated using a van Deemter analysis. Separation power, the product of electric field and residence time, is presented as a parameter for predicting the position of sample streams and for comparing separations under different conditions. Band broadening in [micro]-FFE is governed by diffusion at lower linear velocities and a migration distance-dependent mechanism at higher linear velocities. At higher linear velocities, the parabolic flow profile is elongated, generating a distribution of analyte residence times in the separation channel. This distribution of residence times gives rise to a distribution of migration distances in the lateral direction since analytes spend different amounts of time in the electric field. Equations were derived to predict the effect of electric field and buffer flow rate on broadening. Experimental data were collected to determine whether the derived equations were useful in explaining broadening caused by diffusion and hydrodynamic flow at different linear velocities and electric fields. Overall there was an excellent correlation between the predicted and experimentally observed values allowing linear velocity and electric field to be optimized. Suppression of electroosmotic flow is proposed as a means of reducing [micro]-FFE band broadening due to hydrodynamic effects and maximizing resolution and peak capacity.
- Published
- 2006
11. Using channel depth to isolate and control flow in a micro free-flow electrophoresis device
- Author
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Fonslow, Bryan R., Barocas, Victor H., and Bowser, Michael T.
- Subjects
Electrolysis -- Analysis ,Chemistry - Abstract
A multiple-depth micro flee-flow electrophoresis chip ([micro]-FFE) has been fabricated with a 20-[micro]m-deep separation channel and 78-[micro]m-deep electrode channels. Due to the difference in channel heights, the linear velocity of buffer in the electrode channels is ~15 times that of the buffer in the separation channel. Previous [micro]-FFE devices have been limited by electrolysis product formation at the electrodes. These electrolysis products, manifested as bubbles, decreased the electric field and disrupted the buffer flow profile, limiting performance and preventing continuous operation. Using channel depth to control buffer flow over the electrodes and in the separation channel effectively removes electrolysis products, allowing continuous operation. The linear velocities in the channels were confirmed using particle velocimetry and compared well with values predicted using lubrication theory. A separation potential of 645 V could be applied before significant Joule heating was observed. This corresponded to an electric field of 586 V/cm in the separation channel, a 4-fold increase over our previous design. A separation of fluorescent standards was demonstrated using the new [micro]-FFE device. Resolution increased by a factor of 1.3 over our previous design, even when operated under similar conditions, suggesting that effective removal of electrolysis products is more important than originally thought.
- Published
- 2006
12. Free-flow electrophoresis on an anodic bonded glass microchip
- Author
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Fonslow, Bryan R. and Bowser, Michael T.
- Subjects
Electrophoresis -- Research ,Integrated circuits -- Research ,Integrated circuits -- Design and construction ,Semiconductor chips -- Research ,Semiconductor chips -- Design and construction ,Standard IC ,Chemistry - Abstract
A micro-free-flow electrophoresis chip has been fabricated into a glass wafer etched with 20-[micro]m-deep channels. Wafers were bonded anodically using an intermediate amorphous silicon film. Electric fields as high as 283 V/cm were applied across the separation channel to obtain baseline resolution of fluorescent standards in 4.8 s. The effect of electric fields ranging from 0 to 283 V/cm on the separations and resulting resolutions were examined. Resolution was shown to increase linearly with the applied electric field. Joule heating was not significant under the conditions tested. Instead, the generation of electrolysis products at higher currents proved to be the limiting factor preventing higher separation potentials from being used.
- Published
- 2005
13. Digestion and depletion of abundant proteins improves proteomic coverage
- Author
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Fonslow, Bryan R, Stein, Benjamin D, Webb, Kristofor J, Xu, Tao, Choi, Jeong, Park, Sung Kyu, and Yates, III, John R
- Published
- 2013
- Full Text
- View/download PDF
14. Improving the comprehensiveness and sensitivity of sheathless CE-MS/MS for proteomic analysis
- Author
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Wang, Yueju, Fonslow, Bryan R., Wong, Catherine C.L., Nakorchevsky, Aleksey, and Yates, John R.
- Subjects
Proteomics ,Pyrococcus furiosus ,Isotachophoresis ,Tandem Mass Spectrometry ,Archaeal Proteins ,Escherichia coli Proteins ,Escherichia coli ,Animals ,Electrophoresis, Capillary ,Proteins ,Sensitivity and Specificity ,Article ,Solid Phase Microextraction - Abstract
We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) procedure which employs a high sensitivity porous electrospray ionization (ESI) sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency, we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity.
- Published
- 2012
15. Protein Analysis by Shotgun/Bottom-up Proteomics.
- Author
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Yaoyang Zhang, Fonslow, Bryan R., Bing Shan, Baek, Moon-Chang, and Yates III, John R.
- Subjects
- *
PROTEOMICS , *PROTEIN analysis , *TWO-dimensional electrophoresis , *POLYACRYLAMIDE gel electrophoresis , *CHROMATOGRAPHIC analysis , *POST-translational modification , *MASS spectrometry - Abstract
The article discusses the technique of shotgun/ bottom-up proteomics for the analysis of protein. It states that bottom-up process characterizes protein by analyzing the peptides released from the protein through proteolysis. It discusses several techniques related to shotgun proteomics which include two dimensional polyacrylamide gel electrophoresis (20-PAGE), chromatographic methods and post-translational modification (PTM) analysis by Mass Spectrometry.
- Published
- 2013
- Full Text
- View/download PDF
16. Sequential primed kinases create a damage-responsive phosphodegron on Eco1.
- Author
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Lyons, Nicholas A, Fonslow, Bryan R, Diedrich, Jolene K, Yates, John R, and Morgan, David O
- Subjects
- *
SISTER chromatid exchange , *COHESINS , *PHOSPHORYLATION , *UBIQUITINATION , *NUCLEOTIDE sequence , *HOMOLOGY (Biology) , *DNA damage , *UBIQUITIN - Abstract
Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCFCdc4. We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
17. Improving the Comprehensiveness and Sensitivity of Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry for Proteomic Analysis.
- Author
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Yueju Wang, Fonslow, Bryan R., Wong, Catherine C. L., Nakorchevsky, Aleksey, and John R. Yates , III
- Subjects
- *
SOLID phase extraction , *PROTEOMICS , *CAPILLARY electrophoresis , *TANDEM mass spectrometry , *SEPARATION (Technology) , *PYROCOCCUS furiosus - Abstract
We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) procedure which employs a high sensitivity porous electrospray ionization (ESI) sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency, we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
18. Meta-analysis of global metabolomics and proteomics data to link alterations with phenotype.
- Author
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Patti, Gary J., Tautenhahn, Ralf, Fonslow, Bryan R., Cho, Yonghoon, Deutschbauer, Adam, Arkin, Adam, Northen, Trent, and Siuzdak, Gary
- Subjects
PROTEOMICS ,PHENOTYPES ,CELL metabolism ,BIOCHEMISTRY ,BIOMOLECULES ,FUNCTIONAL genomics ,META-analysis - Abstract
Global metabolomics has emerged as a powerful tool to interrogate cellular biochemistry at the systems level by tracking alterations in the levels of small molecules. One approach to define cellular dynamics with respect to this dysregulation of small molecules has been to consider metabolic flux as a function of time. While flux measurements have proven effective for model organisms, acquiring multiple time points at appropriate temporal intervals for many sample types (e.g., clinical specimens) is challenging. As an alternative, meta-analysis provides another strategy for delineating metabolic cause and effect perturbations. That is, the combination of untargeted metabolomic data from multiple pairwise comparisons enables the association of specific changes in small molecules with unique phenotypic alterations. We recently developed metabolomic software called metaXCMS to automate these types of higher order comparisons. Here we discuss the potential of metaXCMS for analyzing proteomic datasets and highlight the biological value of combining meta-results from both metabolomic and proteomic analyses. The combined meta-analysis has the potential to facilitate efforts in functional genomics and the identification of metabolic disruptions related to disease pathogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
19. Capillary electrophoresis applied to proteomic analysis.
- Author
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Fonslow, Bryan R. and Yates III, John R.
- Published
- 2009
- Full Text
- View/download PDF
20. Addendum: Digestion and depletion of abundant proteins improves proteomic coverage.
- Author
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Fonslow, Bryan R, Stein, Benjamin D, Webb, Kristofor J, Xu, Tao, Choi, Jeong, Park, Sung Kyu, and Yates, John R
- Subjects
- *
DIGESTION , *PROTEOMICS , *PROTEINS , *PROTEOLYTIC enzymes , *SUBSTRATES (Materials science) - Abstract
The article focuses on the use of digestion and depletion of abundant proteins that helps in improving proteomic coverage. It focuses on the detection of low-abundance proteins by decreasing the proteolytic background of sampled peptides from high-abundance proteins. Information on Michaelis-Menten competitive-substrate model is presented.
- Published
- 2014
- Full Text
- View/download PDF
21. Spindle-E cycling between nuage and cytoplasm is controlled by Qin and PIWI proteins.
- Author
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Andress, Arlise, Yanxia Bei, Fonslow, Bryan R., Giri, Ritika, Yilong Wu, Yates, John R., and Carthew, Richard W.
- Subjects
- *
TRANSPOSONS , *SPINDLE apparatus , *PIWI genes , *CYTOPLASM , *GENE expression , *RNA-protein interactions , *NUCLEIC acid amplification techniques - Abstract
Transposable elements (TEs) are silenced in germ cells by a mechanism in which PIWI proteins generate and use PIWIinteracting ribonucleic acid (piRNA) to repress expression of TE genes. piRNA biogenesis occurs by an amplification cycle in microscopic organelles called nuage granules, which are localized to the outer face of the nuclear envelope. One cofactor required for amplification is the helicase Spindle-E (Spn-E). We found that the Spn-E protein physically associates with the Tudor domain protein Qin and the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3). Spn-E and Qin proteins are mutually dependent for their exit from nuage granules, whereas Spn-E and both Aub and Ago3 are mutually dependent for their entry or retention in nuage. The result is a dynamic cycling of Spn-E and its associated factors in and out of nuage granules. This implies that nuage granules can be considered to be hubs for active, mobile, and transient complexes. We suggest that this is in some way coupled with the execution of the piRNA amplification cycle. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
22. Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex.
- Author
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Tien, Jerry F., Umbreit, Neil T., Zelter, Alex, Rifle, Michael, Hoopmann, Michael R., Johnson, Richard S., Fonslow, Bryan R., Yates III, John R., MacCoss, Michael J., Moritz, Robert L., Asbury, Charles L., and Davis, Trisha N.
- Subjects
- *
GENETIC research , *CHROMOSOMES , *MICROTUBULES , *KINETOCHORE , *CHROMATIDS , *SACCHAROMYCES cerevisiae - Abstract
Accurate transmission of genetic material relies on the coupling of chromosomes to spindle microtubules by kinetochores. These linkages are regulated by the conserved Aurora B/Ipl1 kinase to ensure that sister chromatids are properly attached to spindle microtubules. Kinetochore-microtubule attachments require the essential Ndc80 complex, which contains two globular ends linked by large coiled-coil domains. In this study, we isolated a novel ndc80 mutant in Saccharomyces cerevisiae that contains mutations in the coiled-coil domain. This ndc80 mutant accumulates erroneous kinetochore-microtubule attachments, resulting in misalignment of kinetochores on the mitotic spindle. Genetic analyses with suppressors of the ndc80 mutant and in vitro cross-linking experiments suggest that the kinetochore misalignment in vivo stems from a defect in the ability of the Ndc80 complex to stably fold at a hinge in the coiled coil. Previous studies proposed that the Ndc80 complex can exist in multiple conformations: elongated during metaphase and bent during anaphase. However, the distinct functions of individual conformations in vivo are unknown. Here, our analysis revealed a tightly folded conformation of the Ndc80 complex that is likely required early in mitosis. This conformation is mediated by a direct, intracomplex interaction and involves a greater degree of folding than the bent form of the complex at anaphase. Furthermore, our results suggest that this conformation is functionally important in vivo for efficient error correction by Aurora B/Ipl1 and, consequently, to ensure proper kinetochore alignment early in mitosis. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
23. The protein kinase MBK-1 contributes to lifespan extension in daf-2 mutant and germline-deficient Caenorhabditis elegans .
- Author
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Mack HID, Zhang P, Fonslow BR, and Yates JR
- Subjects
- Animals, Animals, Genetically Modified, Caenorhabditis elegans genetics, Caenorhabditis elegans growth & development, Caenorhabditis elegans Proteins metabolism, Forkhead Transcription Factors genetics, Forkhead Transcription Factors metabolism, Gene Expression Regulation, Genotype, Phenotype, Phosphorylation, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Protein-Tyrosine Kinases genetics, Receptor, Insulin metabolism, Receptors, Notch genetics, Receptors, Notch metabolism, Time Factors, Caenorhabditis elegans enzymology, Caenorhabditis elegans Proteins genetics, Longevity genetics, Mutation, Protein-Tyrosine Kinases metabolism, Receptor, Insulin genetics
- Abstract
In Caenorhabditis elegans , reduction of insulin/IGF-1 like signaling and loss of germline stem cells both increase lifespan by activating the conserved transcription factor DAF-16 (FOXO). While the mechanisms that regulate DAF-16 nuclear localization in response to insulin/IGF-1 like signaling are well characterized, the molecular pathways that act in parallel to regulate DAF-16 transcriptional activity, and the pathways that couple DAF-16 activity to germline status, are not fully understood at present. Here, we report that inactivation of MBK-1, the C. elegans ortholog of the human FOXO1-kinase DYRK1A substantially shortens the prolonged lifespan of daf-2 and glp-1 mutant animals while decreasing wild-type lifespan to a lesser extent. On the other hand, lifespan-reduction by mutation of the MBK-1-related kinase HPK-1 was not preferential for long-lived mutants. Interestingly, mbk-1 loss still allowed for DAF-16 nuclear accumulation but reduced expression of certain DAF-16 target genes in germline-less, but not in daf-2 mutant animals. These findings indicate that mbk-1 and daf-16 functionally interact in the germline- but not in the daf-2 pathway. Together, our data suggest mbk-1 as a novel regulator of C. elegans longevity upon both, germline ablation and DAF-2 inhibition, and provide evidence for mbk-1 regulating DAF-16 activity in germline-deficient animals.
- Published
- 2017
- Full Text
- View/download PDF
24. Mass spectrometry-based shotgun proteomic analysis of C. elegans protein complexes.
- Author
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Fonslow BR, Moresco JJ, Tu PG, Aalto AP, Pasquinelli AE, Dillin AG, and Yates JR 3rd
- Subjects
- Animals, Caenorhabditis elegans Proteins metabolism, Immunoprecipitation, Mass Spectrometry, Multiprotein Complexes, Protein Processing, Post-Translational, Proteomics methods, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins genetics, Proteome
- Abstract
Mass spectrometry (MS)-based shotgun proteomics is an enabling technology for the study of C. elegans proteins. When coupled with co-immunoprecipitation (CoIP), new interactions and functions among proteins can be discovered. We provide a general background on protein complexes and methods for their analysis, along with the lifecycle and interaction types of proteins that ultimately define the identifiable components of protein complexes. We highlight traditional biochemical methods to evaluate whether the complexes are sufficiently pure and abundant for analysis with shotgun proteomics. We present two CoIP-MS case studies of protein complexes from C. elegans, using both endogenous and fusion protein antibodies to illustrate the important aspects of their analyses. We discuss results from mass spectrometers with differences in mass accuracy and resolution, along with the relevant information that can be extracted from the data generated, such as protein relative abundance, post-translational modifications, and identification confidence. Finally, we illustrate how comparative analysis can reveal candidate binding partners for biological follow-up and validation. This chapter should act as a complement and extension to the WormBook chapter Biochemistry and molecular biology, which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry.
- Published
- 2014
- Full Text
- View/download PDF
25. Protein analysis by shotgun/bottom-up proteomics.
- Author
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Zhang Y, Fonslow BR, Shan B, Baek MC, and Yates JR 3rd
- Subjects
- Chromatography, High Pressure Liquid, Computational Biology, Electrophoresis, Gel, Two-Dimensional, Isotope Labeling, Peptide Hydrolases metabolism, Protein Processing, Post-Translational, Proteins isolation & purification, Proteins metabolism, Proteins analysis, Proteomics, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Published
- 2013
- Full Text
- View/download PDF
26. Improving the comprehensiveness and sensitivity of sheathless capillary electrophoresis-tandem mass spectrometry for proteomic analysis.
- Author
-
Wang Y, Fonslow BR, Wong CC, Nakorchevsky A, and Yates JR 3rd
- Subjects
- Animals, Archaeal Proteins chemistry, Archaeal Proteins isolation & purification, Escherichia coli chemistry, Escherichia coli Proteins analysis, Escherichia coli Proteins isolation & purification, Isotachophoresis methods, Proteins isolation & purification, Pyrococcus furiosus chemistry, Sensitivity and Specificity, Solid Phase Microextraction methods, Electrophoresis, Capillary methods, Proteins analysis, Proteomics methods, Tandem Mass Spectrometry methods
- Abstract
We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) procedure which employs a high sensitivity porous electrospray ionization (ESI) sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency, we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity.
- Published
- 2012
- Full Text
- View/download PDF
27. Single-step inline hydroxyapatite enrichment facilitates identification and quantitation of phosphopeptides from mass-limited proteomes with MudPIT.
- Author
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Fonslow BR, Niessen SM, Singh M, Wong CC, Xu T, Carvalho PC, Choi J, Park SK, and Yates JR 3rd
- Subjects
- Acids chemistry, Amino Acid Motifs, Amino Acid Sequence, Amygdala chemistry, Animals, Binding Sites, Calcium chemistry, Chemical Precipitation, Consensus Sequence, Female, Mice, Mice, Inbred C57BL, Molecular Sequence Annotation, Molecular Sequence Data, Molecular Weight, Nitrogen Isotopes administration & dosage, Nitrogen Isotopes chemistry, Phosphopeptides chemistry, Phosphorylation, Protein Folding, Proteome chemistry, Proteome isolation & purification, Proteomics instrumentation, Proteomics standards, Sensitivity and Specificity, Solubility, Durapatite chemistry, Phosphopeptides isolation & purification, Proteomics methods
- Abstract
Herein we report the characterization and optimization of single-step inline enrichment of phosphopeptides directly from small amounts of whole cell and tissue lysates (100-500 μg) using a hydroxyapatite (HAP) microcolumn and Multidimensional Protein Identification Technology (MudPIT). In comparison to a triplicate HILIC-IMAC phosphopeptide enrichment study, ∼80% of the phosphopeptides identified using HAP-MudPIT were unique. Similarly, analysis of the consensus phosphorylation motifs between the two enrichment methods illustrates the complementarity of calcium- and iron-based enrichment methods and the higher sensitivity and selectivity of HAP-MudPIT for acidic motifs. We demonstrate how the identification of more multiply phosphorylated peptides from HAP-MudPIT can be used to quantify phosphorylation cooperativity. Through optimization of HAP-MudPIT on a whole cell lysate we routinely achieved identification and quantification of ca. 1000 phosphopeptides from a ∼1 h enrichment and 12 h MudPIT analysis on small quantities of material. Finally, we applied this optimized method to identify phosphorylation sites from a mass-limited mouse brain region, the amygdala (200-500 μg), identifying up to 4000 phosphopeptides per run.
- Published
- 2012
- Full Text
- View/download PDF
28. Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT.
- Author
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Fonslow BR, Carvalho PC, Academia K, Freeby S, Xu T, Nakorchevsky A, Paulus A, and Yates JR 3rd
- Subjects
- Chromatography, Liquid, Electrophoresis, Gel, Two-Dimensional, HeLa Cells, Humans, Tandem Mass Spectrometry, Proteomics
- Abstract
Ideally, shotgun proteomics would facilitate the identification of an entire proteome with 100% protein sequence coverage. In reality, the large dynamic range and complexity of cellular proteomes results in oversampling of abundant proteins, while peptides from low abundance proteins are undersampled or remain undetected. We tested the proteome equalization technology, ProteoMiner, in conjunction with Multidimensional Protein Identification Technology (MudPIT) to determine how the equalization of protein dynamic range could improve shotgun proteomics methods for the analysis of cellular proteomes. Our results suggest low abundance protein identifications were improved by two mechanisms: (1) depletion of high abundance proteins freed ion trap sampling space usually occupied by high abundance peptides and (2) enrichment of low abundance proteins increased the probability of sampling their corresponding more abundant peptides. Both mechanisms also contributed to dramatic increases in the quantity of peptides identified and the quality of MS/MS spectra acquired due to increases in precursor intensity of peptides from low abundance proteins. From our large data set of identified proteins, we categorized the dominant physicochemical factors that facilitate proteome equalization with a hexapeptide library. These results illustrate that equalization of the dynamic range of the cellular proteome is a promising methodology to improve low abundance protein identification confidence, reproducibility, and sequence coverage in shotgun proteomics experiments, opening a new avenue of research for improving proteome coverage.
- Published
- 2011
- Full Text
- View/download PDF
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