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FOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and major determinants of aging in organisms ranging from worms to man. The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response, and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity.

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 SCF(Cdc4). 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.

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

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

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

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.

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.

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.

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.

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.

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]