A century of exercise physiology: key concepts in regulation of glycogen metabolism in skeletal muscle.

Glycogen is a branched, glucose polymer and the storage form of glucose in cells. Glycogen has traditionally been viewed as a key substrate for muscle ATP production during conditions of high energy demand and considered to be limiting for work capacity and force generation under defined conditions. Glycogenolysis is catalyzed by phosphorylase, while glycogenesis is catalyzed by glycogen synthase. For many years, it was believed that a primer was required for de novo glycogen synthesis and the protein considered responsible for this process was ultimately discovered and named glycogenin. However, the subsequent observation of glycogen storage in the absence of functional glycogenin raises questions about the true role of the protein. In resting muscle, phosphorylase is generally considered to be present in two forms: non-phosphorylated and inactive (phosphorylase b) and phosphorylated and constitutively active (phosphorylase a). Initially, it was believed that activation of phosphorylase during intense muscle contraction was primarily accounted for by phosphorylation of phosphorylase b (activated by increases in AMP) to a, and that glycogen synthesis during recovery from exercise occurred solely through mechanisms controlled by glucose transport and glycogen synthase. However, it now appears that these views require modifications. Moreover, the traditional roles of glycogen in muscle function have been extended in recent years and in some instances, the original concepts have undergone revision. Thus, despite the extensive amount of knowledge accrued during the past 100 years, several critical questions remain regarding the regulation of glycogen metabolism and its role in living muscle.

Thermal denaturation and aggregation of apoform of glycogen phosphorylase b. Effect of crowding agents and chaperones.

The effect of protein and chemical chaperones and crowders on thermal stability and aggregation of apoform of rabbit muscle glycogen phosphorylase b (apoPhb) has been studied at 37°C. Proline suppressed heat-induced loss in ability of apoPhb to reconstitution at 37°C, whereas α-crystallin did not reveal a protective action. To compare the antiaggregation activity of intact and crosslinked α-crystallins, an adsorption capacity (AC) of a protein chaperone with respect to a target protein was estimated. This parameter is a measure of the antiaggregation activity. Crosslinking of α-crystallin results in 11-fold decrease in the initial AC. The nonlinear character of the relative initial rate of apoPhb aggregation versus the [intact α-crystallin]/[apoPhb] ratio plot is indicative of the decrease in the AC of α-crystallin with increasing the [α-crystallin]/[apoPhb] ratio and can be interpreted as an evidence for dynamic chaperone structure and polydispersity of α-crystallin-target protein complexes. As for chemical chaperones, a semisaturation concentration of the latter was used as a characteristic of the antiaggregation activity. A decrease in the semisaturation concentration for proline was observed in the presence of the crowders (polyethylene glycol and Ficoll-70).

From protein complexes to subunit backbone fragments: a multi-stage approach to native mass spectrometry.

Native mass spectrometry (MS) is becoming an important integral part of structural proteomics and system biology research. The approach holds great promise for elucidating higher levels of protein structure: from primary to quaternary. This requires the most efficient use of tandem MS, which is the cornerstone of MS-based approaches. In this work, we advance a two-step fragmentation approach, or (pseudo)-MS(3), from native protein complexes to a set of constituent fragment ions. Using an efficient desolvation approach and quadrupole selection in the extended mass-to-charge (m/z) range, we have accomplished sequential dissociation of large protein complexes, such as phosporylase B (194 kDa), pyruvate kinase (232 kDa), and GroEL (801 kDa), to highly charged monomers which were then dissociated to a set of multiply charged fragmentation products. Fragment ion signals were acquired with a high resolution, high mass accuracy Orbitrap instrument that enabled highly confident identifications of the precursor monomer subunits. The developed approach is expected to enable characterization of stoichiometry and composition of endogenous native protein complexes at an unprecedented level of detail.

Alkylated trihydroxyacetophenone as a MALDI matrix for hydrophobic peptides.

Hydrophobic peptides are difficult to detect in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), because of the hydrophilic properties of conventional matrices and the low affinity for hydrophobic peptides. Recently, we reported on alkylated dihydroxybenzoic acid (ADHB) as a matrix additive for hydrophobic peptides; however, the peptides were detected in the rim of the matrix-analyte dried spot. Here, we report on a novel matrix, alkylated trihydroxyacetophenone (ATHAP), which is a 2,4,6-trihydroxyacetophenone derivative incorporating a hydrophobic alkyl chain on the acetyl group and thus is expected to have an affinity for hydrophobic peptides. ATHAP increased the sensitivity of hydrophobic peptides 10-fold compared with α-cyano-4-hydroxycinnamic acid (CHCA), in which the detection of hydrophilic peptides was suppressed. The peptides were detected throughout the entire matrix-analyte dried spot using ATHAP, overcoming the difficulty of finding a "sweet spot" when using ADHB. In addition, ATHAP functioned alone as a matrix, unlike ADHB as an additive. In phosphorylase b digests analysis, hydrophobic peptides, which were not detected with CHCA for 1 pmol, were detected with this matrix, confirming that ATHAP led to increased sequence coverage and may extend the range of target analytes in MALDI-MS.

Determination of ¹5N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

Alkylated dihydroxybenzoic acid as a MALDI matrix additive for hydrophobic peptide analysis.

Hydrophobic peptides are generally difficult to detect using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) because the majority of MALDI matrixes are hydrophilic and therefore have a low affinity for hydrophobic peptides. Here, we report on a novel matrix additive, o-alkylated dihydroxybenzoic acid (ADHB), which is a 2,5-dihydroxybenzoic acid (DHB) derivative incorporating a hydrophobic alkyl chain on a hydroxyl group to improve its affinity for hydrophobic peptides, thereby improving MALDI-MS sensitivity. The addition of ADHB to the conventional matrix α-cyano-4-hydroxycinnamic acid (CHCA) improved the sensitivity of hydrophobic peptides 10- to 100-fold. The sequence coverage of phosphorylase b digest was increased using ADHB. MS imaging indicated that hydrophobic peptides were enriched in the rim of a matrix/analyte dried spot when ADHB was used. In conclusion, the addition of ADHB to the standard matrix led to improved sensitivity of hydrophobic peptides by MALDI-MS.

Size exclusion chromatography with multi detection in combination with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry as a tool for unraveling the mechanism of the enzymatic polymerization of polysaccharides.

Determination of the size distributions of natural polysaccharides is a challenging task. More advantageous for characterization are well-defined synthetic (hyper)-branched polymers. In this study we concentrated on synthetic amylopectin analogues in order to obtain and compare all available data for different distributions and size dependence of molecular weights. Two groups of well-defined synthetic branched polysaccharides were synthesized via an in vitro enzyme-catalyzed reaction using the enzyme phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme. Synthetic polymers had a tunable degree of branching (2%-13% determined via (1)H NMR) and a tunable degree of polymerization (30-350 determined indirectly via UV spectrometry). The systems used for separation and characterization of branched polysaccharides were SEC-DMSO/LiBr and multi detection (refractive index detector, viscosity detector, and multi angle light scattering detector) and SEC-water/0.02% NaN(3); and SEC-50 mM NaNO(3)/0.02% NaN(3) and multi detection. Additionally the side chain length distribution of enzymatically debranched polysaccharides was investigated by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. With this combination of characterization techniques, we were able not only to characterize the amylopectin analogues but also to solve parts of the molecular mechanism of their enzymatic polymerization. Moreover our materials showed potential to be standards in the field of natural polysaccharides characterization.

Combined action of chemical chaperones on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b.

Chemical chaperones are a class of small molecules, which enhance protein stability, folding, inhibit protein aggregation, and are used for long-term storage of therapeutic proteins. The combined action of chemical chaperones trehalose, betaine and lysine on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b (Phb) has been studied. Dynamic light scattering data indicate that the affinity of trehalose to Phb increased in the presence of betaine or lysine at both stages (stage of nucleation and aggregate growth) of enzyme aggregation at 48 °C, in contrast, the affinity of betaine to the enzyme in the presence of lysine remained practically unchanged. According to differential scanning calorimetry and analytical ultracentrifugation data, the mixture of trehalose and betaine stabilized Phb stronger than either of them in total. Moreover, the destabilizing effect of lysine on the enzyme was almost completely compensated by trehalose and only partially by betaine. The main protective effect of the mixtures of osmolytes and lysine is associated with their influence on the dissociation/denaturation stage, which is the rate-limiting one of Phb aggregation. Thus, a pair of chaperones affects the stability, oligomeric state, and aggregation of Phb differently than individual chaperones.

Does the crowded cell-like environment reduce the chaperone-like activity of α-crystallin?

The effect of crowding on the chaperone-like activity of α-crystallin has been studied using aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle as an aggregation test system. The merit of this test system is the possibility of testing agents that directly affect the stage of aggregation of the protein molecules. It was shown that the solution of Phb denatured by UV contained aggregates with a hydrodynamic radius of 10.4 nm. These aggregates are relatively stable at 20 °C; however, they reveal a tendency to stick further in the presence of crowding agents. The study of the effect of α-crystallin on the aggregation of UV-irradiated Phb in the presence of the crowding agents by dynamic light scattering at 37 °C showed that under crowding conditions the antiaggregation ability of α-crystallin was weakened. On the basis of the analytical ultracentrifugation, size-exclusion chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the scheme of interaction of UV-irradiated Phb and α-crystallin has been proposed. It is assumed that chaperone-target protein complexes of two types are formed, namely, the complexes of dissociated forms of α-crystallin with a protein substrate and high-mass α-crystallin-denatured protein complexes. The complexes of the first type reveal a weak propensity to aggregate even under crowding conditions. The complexes of the second type are characterized by the lower rate of aggregation in comparison with that of original UV-irradiated Phb. However, crowding stimulates the rate of aggregation of these complexes, resulting in the above-mentioned decrease in the chaperone-like activity of α-crystallin.

A method for N-terminal de novo sequencing of Nα-blocked proteins by mass spectrometry.

A method for de novo sequencing of N(α)-blocked proteins by mass spectrometry (MS) is presented. The approach consists of enzymatic digestion of N(α)-blocked protein, recovery of N-terminal peptide by depletion of non-N-terminal peptides from the digest pool, and selective derivatization of a C-terminal α-carboxyl group of isolated N-terminal peptide. The C-terminal α-carboxyl group of the N-terminal peptide was selectively derivatized with 3-aminopropyl-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine), according to oxazolone chemistry. The reagent TMPP-propylamine was designed to facilitate sequence analysis with MALDI-MS by mass- and charge-tagging. All of the identities and N-terminal sequences of two N(α)-acetylated proteins (rabbit phosphorylase b and bovine calmodulin) and human orexin A, which has pyroglutamic acid at the N-terminus, were successfully analyzed by allowing for the y-type ions almost exclusively.

Common phenotype of resting mouse extensor digitorum longus and soleus muscles: equal ATPase and glycolytic flux during transient anoxia.

Rates of ATPase and glycolysis are several times faster in actively contracting mouse extensor digitorum longus muscle (EDL) than soleus (SOL), but we find these rates are not distinguishable at rest. We used a transient anoxic perturbation of steady state energy balance to decrease phosphocreatine (PCr) reversibly and to measure the rates of ATPase and of lactate production without muscle activation or contraction. The rate of glycolytic ATP synthesis is less than the ATPase rate, accounting for the continual PCr decrease during anoxia in both muscles. We fitted a mathematical model validated with properties of enzymes and solutes measured in vitro and appropriate for the transient perturbation of these muscles to experimental data to test whether the model accounts for the results. Simulations showed equal rates of ATPase and lactate production in both muscles. ATPase controls glycolytic flux by feedback from its products. Adenylate kinase function is critical because a rise in [AMP] is necessary to activate glycogen phosphorylase. ATPase is the primary source of H+ production. The sum of contributions of the 13 reactions of the glycogenolytic and glycolytic network to total proton load is negligible. The stoichiometry of lactate and H+ production is near unity. These results identify a default state of energy metabolism for resting muscle in which there is no difference in the metabolic phenotype of EDL and SOL. Therefore, additional control mechanisms, involving higher ATPase flux and [Ca2+], must exist to explain the well-known difference in glycolytic rates in fast-twitch and slow-twitch muscles in actively contracting muscle.

An adsorption-based model for pulse duration in resistive-pulse protein sensing.

We have been investigating an electrochemical single-molecule counting experiment called nanopore resistive-pulse sensing. The sensor element is a conically shaped gold nanotube embedded in a thin polymeric membrane. We have been especially interested in counting protein molecules using these nanotube sensors. This is accomplished by placing the nanotube membrane between two electrolyte solutions, applying a transmembrane potential difference, and measuring the resulting ionic current flowing through the nanopore. In simplest terms, when a protein molecule enters and translocates the nanopore, it transiently blocks the ion current, resulting in a downward current pulse. We have found that the duration of such current-pulses are many orders of magnitude longer than the electrophoretic transport time of the protein through the nanotube detection zone. We develop here a simple model that accounts for this key, and previously explained, observation. This model assumes that the protein molecule engages in repeated adsorption/desorption events to/from the nanotube walls as it translocates through the detection zone. This model not only accounts for the long pulse duration but also for the triangular shape of the current pulse and the increase in the standard deviation of the pulse duration with increasing protein size. Furthermore, the results of our analyses are in general agreement with results obtained from other investigations of protein adsorption to surfaces. This includes the observations that smaller proteins stick more readily to the surface but remain adsorbed for shorter times than larger proteins. In addition, the sticking probabilities calculated from our data are in general agreement with results obtained from other methods.

1-(3-Deoxy-3-fluoro-beta-d-glucopyranosyl) pyrimidine derivatives as inhibitors of glycogen phosphorylase b: Kinetic, crystallographic and modelling studies.

Design of inhibitors of glycogen phosphorylase (GP) with pharmaceutical applications in improving glycaemic control in type 2 diabetes is a promising therapeutic strategy. The catalytic site of muscle glycogen phosphorylase b (GPb) has been probed with five deoxy-fluro-glucose derivatives. These inhibitors had fluorine instead of hydroxyl at the 3' position of the glucose moiety and a variety of pyrimidine derivatives at the 1' position. The best of this carbohydrate-based family of five inhibitors displays a K(i) value of 46muM. To elucidate the mechanism of inhibition for these compounds, the crystal structures of GPb in complex with each ligand were determined and refined to high resolution. The structures demonstrated that the inhibitors bind preferentially at the catalytic site and promote the less active T state conformation of the enzyme by making several favorable contacts with residues of the 280s loop. Fluorine is engaged in hydrogen bond interactions but does not improve glucose potency. The pyrimidine groups are located between residues 284-286 of the 280s loop, Ala383 of the 380s loop, and His341 of the beta-pocket. These interactions appear important in stabilizing the inactive quaternary T state of the enzyme. As a follow up to recent computations performed on beta-d-glucose pyrimidine derivatives, tautomeric forms of ligands 1-5 were considered as potential binding states. Using Glide-XP docking and QM/MM calculations, the ligands 2 and 5 are predicted to bind in different tautomeric states in their respective GPb complexes. Also, using alpha-d-glucose as a benchmark model, a series of substitutions for glucose -OH at the 3' (equatorial) position were investigated for their potential to improve the binding affinity of glucose-based GPb catalytic site inhibitors. Glide-XP and quantum mechanics polarized ligand (QPLD-SP/XP) docking calculations revealed favorable binding at this position to be dominated by hydrogen bond contributions; none of the substitutions (including fluorine) out-performed the native -OH substituent which can act both as hydrogen bond donor and acceptor. The structural analyses of these compounds can be exploited towards the development of better inhibitors.

Ion mobility-mass spectrometry of phosphorylase B ions generated with supercharging reagents but in charge-reducing buffer.

We investigate whether "supercharging" reagents able to shift the charge state distributions (CSDs) of electrosprayed protein ions upward also influence gas-phase protein structure. A differential mobility analyzer and a mass spectrometer are combined in series (DMA-MS) to measure the mass and mobility of monomer and multimeric phosphorylase B ions (monomer molecular weight ∼97 kDa) in atmospheric pressure air. Proteins are electrosprayed from charge-reducing triethylammonium formate in water (pH = 6.8) with and without the addition of the supercharging reagent tetramethylene sulfone (sulfolane). Because the DMA measures ion mobility prior to collisional heating or declustering, it probes the structure of supercharged protein ions immediately following solvent (water) evaporation. As in prior studies, the addition of sulfolane is found to drastically increase both the mean and maximum charge state of phosphorylase B ions. Ions from all protein n-mers were found to yield mobilities that, for a given charge state, were ∼6-10% higher in the absence of sulfolane. We find that the mobility decrease which arises with sulfolane is substantially smaller than that typically observed for folded-to-unfolded transitions in protein ions (where a ∼60% decrease in mobility is typical), suggesting that supercharging reagents do not cause structural protein modifications in solution as large as noted recently by Williams and colleagues [E. R. Williams et al., J. Am. Soc. Mass Spectrom., 2010, 21, 1762-1774]. In fact, the measurements described here indicate that the modest mobility decrease observed can be partly attributed to sulfolane trapping within the protein ions during DMA measurements, and probably also in solution. As the most abundant peaks in measured mass-mobility spectra for ions produced with and without sulfolane correspond to non-covalently bound phosphorylase B dimers, we find that in spite of a change in mobility/cross section, sulfolane addition does not substantially alter the structure of non-covalently bound protein complexes in the gas-phase.

Improving Peptide Fragmentation for Hydrogen-Deuterium Exchange Mass Spectrometry Using a Time-Dependent Collision Energy Calculator.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is becoming a popular technique for interrogating biological systems. In recent years, advancements have been made to increase peptide coverage for proteins that resist digestion such as antibodies and membrane proteins. These methods commonly include using alternative digestion enzymes or longer chromatographic gradients, which may be expensive or time-consuming to implement. Here, we recommend an efficient proteomics-based approach to increase peptide confidence and coverage. A major filtering parameter for peptides in HDX is the number of product ions detected; this is a result of the collision energy (CE) applied within the MS. A traditional linear ramp achieves optimal CE for only short periods of time. More product ions will be created and detected if optimal CE can be achieved for a longer period of time. As a result, the coverage, redundancy, and data confidence are all increased. We achieved this by implementing a mobility-dependent CE look up table (LUT) which increases the CE as a function of mobility. We developed a program to calculate the optimal CE for a set of peptides and MS settings based on initial reference samples. We demonstrated the utility of the CE LUT on three protein samples including the soluble phosphorylase B, IgG2, and the membrane-stabilized AcrB. We showed that applying a CE LUT provided 8.5-50% more peptides compared to a linear CE ramp. The results demonstrate that a time-dependent CE LUT is a quick and inexpensive method to increase data confidence and peptide abundance for HDX-MS experiments.

Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide.

The activity of glycogen phosphorylase, an enzyme that is activated by both cAMP and calcium, was used as an indicator of the state of the cytoplasm after chemotactic stimulation of polymorphonuclear leukocytes (neutrophils). The activity of the enzyme showed a clear dependence on cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a 4-5-fold increase in activity of phosphorylase a. In the absence of external Ca2+, A23187 caused only brief transient activation of phosphorylase; probably reflecting release of sequestered intracellular Ca2+. Addition of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold activation of the enzyme. The dose-dependence of activation by FNLLP showed a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The phosphorylase activity peaks by 90 s and then declines, returning to basal levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with 10(-7) M peptide. This finding suggests that the cells do not need to maintain elevated cytoplasmic calcium levels to exhibit stimulated locomotion. Thus, if calcium continues to modulate the motility, there either must be highly localized changes that are not detected in measures of the total cytoplasm, or the sensitivity to calcium must be variable such that basal levels are sufficient to maintain locomotion. Cells loaded with the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of external Ca2+ had elevated phosphorylase levels before addition of FNLLP. Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it clearly alters some aspects of the neutrophil physiology. Phosphorylase a appears to be a sensitive, nonperturbing indicator of the cytoplasmic calcium levels.

Structural basis for the activation of glycogen phosphorylase b by adenosine monophosphate.

The three-dimensional structure of the activated state of glycogen phosphorylase (GP) as induced by adenosine monophosphate (AMP) has been determined from crystals of pyridoxalpyrophosphoryl-GP. The same quaternary changes relative to the inactive conformation as those induced by phosphorylation are induced by AMP, although the two regulatory signals function through different local structural mechanisms. Moreover, previous descriptions of the phosphorylase active state have been extended by demonstrating that, on activation, the amino- and carboxyl-terminal domains of GP rotate apart by 5 degrees, thereby increasing access of substrates to the catalytic site. The structure also reveals previously unobserved interactions with the nucleotide that accounts for the specificity of the nucleotide binding site for AMP in preference to inosine monophosphate.

Breast cancers utilize hypoxic glycogen stores via PYGB, the brain isoform of glycogen phosphorylase, to promote metastatic phenotypes.

In breast cancer, tumor hypoxia has been linked to poor prognosis and increased metastasis. Hypoxia activates transcriptional programs in cancer cells that lead to increased motility and invasion, as well as various metabolic changes. One of these metabolic changes, an increase in glycogen metabolism, has been further associated with protection from reactive oxygen species damage that may lead to premature senescence. Here we report that breast cancer cells significantly increase glycogen stores in response to hypoxia. We found that knockdown of the brain isoform of an enzyme that catalyzes glycogen breakdown, glycogen phosphorylase B (PYGB), but not the liver isoform, PYGL, inhibited glycogen utilization in estrogen receptor negative and positive breast cancer cells; whereas both independently inhibited glycogen utilization in the normal-like breast epithelial cell line MCF-10A. Functionally, PYGB knockdown and the resulting inhibition of glycogen utilization resulted in significantly decreased wound-healing capability in MCF-7 cells and a decrease in invasive potential of MDA-MB-231 cells. Thus, we identify PYGB as a novel metabolic target with potential applications in the management and/or prevention of metastasis in breast cancer.

Dynamically multiplexed ion mobility time-of-flight mass spectrometry.

Ion mobility spectrometry-time-of-flight mass spectrometry (IMS-TOFMS) has been increasingly used in analysis of complex biological samples. A major challenge is to transform IMS-TOFMS to a high-sensitivity, high-throughput platform, for example, for proteomics applications. In this work, we have developed and integrated three advanced technologies, including efficient ion accumulation in an ion funnel trap prior to IMS separation, multiplexing (MP) of ion packet introduction into the IMS drift tube, and signal detection with an analog-to-digital converter, into the IMS-TOFMS system for the high-throughput analysis of highly complex proteolytic digests of, for example, blood plasma. To better address variable sample complexity, we have developed and rigorously evaluated a novel dynamic MP approach that ensures correlation of the analyzer performance with an ion source function and provides the improved dynamic range and sensitivity throughout the experiment. The MP IMS-TOFMS instrument has been shown to reliably detect peptides at a concentration of 1 nM in the presence of a highly complex matrix, as well as to provide a 3 orders of magnitude dynamic range and a mass measurement accuracy of better than 5 ppm. When matched against human blood plasma database, the detected IMS-TOF features were found to yield approximately 700 unique peptide identifications at a false discovery rate (FDR) of approximately 7.5%. Accounting for IMS information gave rise to a projected FDR of approximately 4%. Signal reproducibility was found to be greater than 80%, while the variations in the number of unique peptide identifications were <15%. A single sample analysis was completed in 15 min that constitutes almost 1 order of magnitude improvement compared to a more conventional LC-MS approach.

[Erythropoietin protects neuron against ketamine induced injuries].

OBJECTIVE: To investigate whether erythropoietin (EPO) protects neuron against ketamine induced injuries. METHODS: Neurons were obtained from SD rat brain, cultured, and treated with ketamine of the concentrations of 0.1, 1, 10, and 30 micromol/L respectively. Neurons not treated by any agent were used as control group. Another neurons were divided into 3 groups undergoing the treatment of ketamine of the terminal concentration of 10 micromol/L, EPO + ketamine group undergoing the treatment of 10 micromol/L ketamine and EPO of the terminal concentrations of 0.3, 1, 3, and 10 U/ml, and ketamine + EPO + LY294002 group undergoing the treatment of 10 micromol/L ketamine, 1 U/ml EPO, and 10 micromol/L LY294002, a P13k inhibitor. Twenty-four hours after the co-inoculation the survival rates of the neurons were detected by MTT method. The apoptotic rate was detected by TUNEL assay. The neuron vitality was measured by MTT assay. Apoptotic neurons were measured by TUNEL assay. The activity of caspase-3 was detected with the caspase-3 fluorometric assay kit. The level of pAkt protein was analyzed by Western blotting. RESULTS: The survival rates of the neurons exposed to ketamine of the concentrations of 1, 10, and 30 micromol/L were (91 +/- 5)%, (42 +/- 6)%, and (23 +/- 7)% respectively, significantly lower than that of the control group (P < 0.05 or P < 0.01). The survival rates of the neurons treated by 10 micromol/L ketamine and EPO of the concentrations of 0.3, 1, 3, and 10 U/ml were (73 +/- 6)%, (86 +/- 9)%, (78 +/- 8)%, and (71 +/- 10)% respectively, all significantly high than that of the 10 micromol/L ketamine group (P < 0.05 or P < 0.01). The number of apoptotic neurons of the 10 micromol/L ketamine group was significantly higher than that pf the control group, the number of apoptotic neurons of the 10 micromol/L ketamine + 10 U/ml EPO group was significantly lower than that of the 10 micromol/L ketamine, and the number of apoptotic neurons of the ketamine + EPO + LY294002 group was (130 +/- 30)%, remarkably lower than that of the ketamine group. (P < 0.01), and the relative activity of caspase-3 of the 10 micromol/L ketamine group was (280 +/- 60)%, significantly higher than that of the control group, (P < 0.01). The relative activity of caspase-3 of the ketamine + EPO + LY294002 group was (220 +/- 34)%, significantly higher than that of the ketamine + EPO (P < 0.01). The pAkt protein level of the 10 micromol/L ketamine group was significantly lower than that of the control group (P < 0.05), the pAkt protein level of the 10 micromol/L ketamine + 10 U/ml EPO group was significantly higher than that of the 10 micromol/L ketamine group (P < 0.01), and the pAkt protein level of the ketamine + EPO + LY294002 group was significantly lower than that of the ketamine + 10 U/ml EPO group (P < 0.01). CONCLUSION: EPO affords significant neuroprotection against ketamine induced injury in neurons via PI3K/Akt-mediated signaling pathway.