Platelet proteome dynamics in hibernating 13-lined ground squirrels.

Hibernating mammals undergo a dramatic drop in temperature and blood flow during torpor, yet avoid stasis blood clotting through mechanisms that remain unspecified. The effects of hibernation on hemostasis are especially complex, as cold temperatures generally activate platelets, resulting in platelet clearance and cold storage lesions in the context of blood transfusion. With a hibernating body temperature of 4°C-8°C, 13-lined ground squirrels (Ictidomys tridecemlineatus) provide a model to study hemostasis as well as platelet cold storage lesion resistance during hibernation. Here, we quantified and systematically compared proteomes of platelets collected from ground squirrels at summer (active), fall (entrance), and winter (topor) to elucidate how molecular-level changes in platelets may support hemostatic adaptations in torpor. Platelets were isolated from a total of 11 squirrels in June, October, and January. Platelet lysates from each animal were digested with trypsin prior to 11-plex tandem mass tag (TMT) labeling, followed by LC-MS/MS analysis for relative protein quantification. We measured >700 proteins with significant variations in abundance in platelets over the course of entrance, torpor, and activity-including systems of proteins regulating translation, secretion, metabolism, complement, and coagulation cascades. We also noted species-specific differences in levels of hemostatic, secretory, and inflammatory regulators in ground squirrel platelets relative to human platelets. Altogether, we provide the first ever proteomic characterization of platelets from hibernating animals, where systematic changes in metabolic, hemostatic, and other proteins may account for physiological adaptations in torpor and also inform translational effort to improve cold storage of human platelets for transfusion.

Phosphoproteomic quantitation and causal analysis reveal pathways in GPVI/ITAM-mediated platelet activation programs.

Platelets engage cues of pending vascular injury through coordinated adhesion, secretion, and aggregation responses. These rapid, progressive changes in platelet form and function are orchestrated downstream of specific receptors on the platelet surface and through intracellular signaling mechanisms that remain systematically undefined. This study brings together cell physiological and phosphoproteomics methods to profile signaling mechanisms downstream of the immunotyrosine activation motif (ITAM) platelet collagen receptor GPVI. Peptide tandem mass tag (TMT) labeling, sample multiplexing, synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) detected >3000 significant (false discovery rate < 0.05) phosphorylation events on >1300 proteins over conditions initiating and progressing GPVI-mediated platelet activation. With literature-guided causal inference tools, >300 site-specific signaling relations were mapped from phosphoproteomics data among key and emerging GPVI effectors (ie, FcRγ, Syk, PLCγ2, PKCδ, DAPP1). Through signaling validation studies and functional screening, other less-characterized targets were also considered within the context of GPVI/ITAM pathways, including Ras/MAPK axis proteins (ie, KSR1, SOS1, STAT1, Hsp27). Highly regulated GPVI/ITAM targets out of context of curated knowledge were also illuminated, including a system of >40 Rab GTPases and associated regulatory proteins, where GPVI-mediated Rab7 S72 phosphorylation and endolysosomal maturation were blocked by TAK1 inhibition. In addition to serving as a model for generating and testing hypotheses from omics datasets, this study puts forth a means to identify hemostatic effectors, biomarkers, and therapeutic targets relevant to thrombosis, vascular inflammation, and other platelet-associated disease states.

p300 and cAMP response element-binding protein-binding protein in skeletal muscle homeostasis, contractile function, and survival.

BACKGROUND: Reversible ε-amino acetylation of lysine residues regulates transcription as well as metabolic flux; however, roles for specific lysine acetyltransferases in skeletal muscle physiology and function are unknown. In this study, we investigated the role of the related acetyltransferases p300 and cAMP response element-binding protein-binding protein (CBP) in skeletal muscle transcriptional homeostasis and physiology in adult mice. METHODS: Mice with skeletal muscle-specific and inducible knockout of p300 and CBP (PCKO) were generated by crossing mice with a tamoxifen-inducible Cre recombinase expressed under the human α-skeletal actin promoter with mice having LoxP sites flanking exon 9 of the Ep300 and Crebbp genes. Knockout of PCKO was induced at 13-15 weeks of age via oral gavage of tamoxifen for 5 days to both PCKO and littermate control [wildtype (WT)] mice. Body composition, food intake, and muscle function were assessed on day 0 (D0) through 5 (D5). Microarray and tandem mass tag mass spectrometry analyses were performed to assess global RNA and protein levels in skeletal muscle of PCKO and WT mice. RESULTS: At D5 after initiating tamoxifen treatment, there was a reduction in body weight (-15%), food intake (-78%), stride length (-46%), and grip strength (-45%) in PCKO compared with WT mice. Additionally, ex vivo contractile function [tetanic tension (kPa)] was severely impaired in PCKO vs. WT mice at D3 (~70-80% lower) and D5 (~80-95% lower) and resulted in lethality within 1 week-a phenotype that is reversed by the presence of a single allele of either p300 or CBP. The impaired muscle function in PCKO mice was paralleled by substantial transcriptional alterations (3310 genes; false discovery rate < 0.1), especially in gene networks central to muscle contraction and structural integrity. This transcriptional uncoupling was accompanied by changes in protein expression patterns indicative of impaired muscle function, albeit to a smaller magnitude (446 proteins; fold-change > 1.25; false discovery rate < 0.1). CONCLUSIONS: These data reveal that p300 and CBP are required for the control and maintenance of contractile function and transcriptional homeostasis in skeletal muscle and, ultimately, organism survival. By extension, modulating p300/CBP function may hold promise for the treatment of disorders characterized by impaired contractile function in humans.

Identification of PKCα-dependent phosphoproteins in mouse retina.

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKCα), the targets of PKCα phosphorylation in the retina have not been identified. PKCα activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKCα antibody and antibodies to phosphorylated PKC motifs. PKCα activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKCα-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKCα knockout (PKCα-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKCα-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906. SIGNIFICANCE: Retinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKCα, though the specific mechanism by which PKCα modulates RBC physiology is unknown. This study examined PKCα phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKCα-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKCα-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKCα-dependent modulation of RBC physiology.

Identification, Quantification, and System Analysis of Protein N-ε Lysine Methylation in Anucleate Blood Platelets.

Protein posttranslational modifications critically regulate a range of physiological and disease processes. In addition to tyrosine, serine, and threonine phosphorylation, reversible N-ε acylation and alkylation of protein lysine residues also modulate diverse aspects of cellular function. Studies of lysine acyl and alkyl modifications have focused on nuclear proteins in epigenetic regulation; however, lysine modifications are also prevalent on cytosolic proteins to serve increasingly apparent, although less understood roles in cell regulation. Here, the methyl-lysine (meK) proteome of anucleate blood platelets is characterized. With high-resolution, multiplex MS methods, 190 mono-, di-, and tri-meK modifications are identified on 150 different platelet proteins-including 28 meK modifications quantified by tandem mass tag (TMT) labeling. In addition to identifying meK modifications on calmodulin (CaM), GRP78 (HSPA5, BiP), and EF1A1 that have been previously characterized in other cell types, more novel modifications are also uncovered on cofilin, drebin-like protein (DBNL, Hip-55), DOCK8, TRIM25, and numerous other cytoplasmic proteins. Together, the results and analyses support roles for lysine methylation in mediating cytoskeletal, translational, secretory, and other cellular processes. MS data for this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012217.

Fast chromatography in the regulated bioanalytical environment: sub-2-µm versus fused-core particles.

Increased throughput and rapid data turnaround have necessitated the need for faster LC separations in the regulated bioanalytical environment; sub-2-µm and fused-core chromatography are two viable approaches to accomplish this. Sub-2-µm columns offer high efficiency and resolution separations at the cost of high system backpressure and are best suited for preclinical sample analysis, whereas fused-core columns offer slightly lower efficiency and resolution, but are more tolerant to dirty extracts (such as those seen in clinical samples) that may clog the inlet filter. For clinical sample analyses, sub-2-µm columns should be restricted to sample extracts obtained via LLE or SLE, whereas fused-core columns can be used with sample preparations techniques ranging from protein precipitation to SLE.

Implementation of high-temperature superficially porous technologies for rapid LC-MS/MS diastereomer bioanalysis.

BACKGROUND: The fast-paced nature of the pharmaceutical industry requires robust bioanalytical methods to endure the high-throughput sample demands of the production environment. RESULTS: A rapid, accurate and precise LC-MS/MS method was developed for the quantitation of a diastereomer quartet in human plasma. Virtually all of the phosphatidylcholine and most of the lysophosphatidylcholine from human plasma were removed using a phospholipid-removing protein precipitation 96-well plate. An Agilent Poroshell SB-C18 2.1 × 50 mm superficially porous column was used at 100°C and 1.2 ml/min to separate a diastereomer quartet in <2.5 min. Peak shape, retention and resolution were maintained over nearly 200 extracted bioanalytical samples under these separation conditions. The method was tested for accuracy and precision; the assay inter-run accuracy and precision were minus 7.2-0.7% and 2.1-11.9%, respectively (n = 18). CONCLUSION: The application of the superficially porous column resulted in twofold response increase and a 2.6-fold reduction in cycle time compared with a 3.5-µm column performing under comparable resolution conditions.

Using temperature to optimize resolution and reduce analysis times for bioanalytical diastereomer LC-MS/MS separations.

A rugged and reproducible liquid chromatographic tandem mass spectrometric bioanalytical method was developed for the quantitation of drug stereoisomers in human plasma. Column temperature was shown to be an important variable toward optimizing diastereomer selectivity, resolution and analysis cycle time. Non-linear Van't Hoff plots and changes in peak shape with temperature suggested that selectivity was governed by multiple retention mechanisms. The high temperature chromatography method was validated and used to analyze samples from human clinical trials. Utilization of high temperature chromatography offered alternative selectivity and is a viable approach for difficult separations in regulated bioanalysis.

A high-throughput LC-MS/MS method for the quantitation of posaconazole in human plasma: Implementing fused core silica liquid chromatography.

A rapid and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the determination of posaconazole concentrations in human plasma was validated. Posaconazole was extracted from human plasma using mixed-mode cation exchange solid phase extraction in a 96-well plate format followed by gradient separation on a fused-core Halo C18 column. The analyte and its corresponding internal standard were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray ionization source operated in the positive ion mode. The calibration range of the method was 5.00-5000ng/mL using a 50microL aliquot of plasma. The assay inter-run accuracy and precision were-4.6-2.8% and 2.3-8.7%, respectively (n=18). The results from method validation indicate the method to be sensitive, selective, accurate, and reproducible. The method was successfully applied to the routine analysis of clinical samples with the fused-core silica columns providing excellent reproducibility for greater than 1000 injections per column.

Fused-core particle technology as an alternative to sub-2-microm particles to achieve high separation efficiency with low backpressure.

Fused-Core particles have recently been introduced as an alternative to using sub-2-microm particles in chromatographic separations. Fused-Core particles are composed of a 1.7 microm solid core surrounded by a 0.5 microm porous silica layer (d(p) = 2.7 microm) to reduce mass transfer and increase peak efficiency. The performance of two commercially available Fused-Core particles (Advanced Materials Technology Halo C18 and Supelco Ascentis Express C18) was compared with sub-2-microm particles from Waters, Agilent, and Thermo Scientific. Although the peak efficiencies were only approximately 80% of those obtained by the Waters Acquity particles, the 50% lower backpressure allowed columns to be coupled in series to increase peak efficiency to 92,750 plates. The low backpressure and high efficiencies of the Fused-Core particles offer a viable alternative to using sub-2-microm particles and very-high-pressure LC instrumentation.

Detection of g protein coupled receptor mediated adenylyl cyclase activity by capillary electrophoresis using fluorescently labeled ATP.

A capillary electrophoresis (CE) laser-induced fluorescence (LIF) assay was developed for the detection of G protein coupled receptor mediated adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, cell membranes coexpressing the stimulatory G protein fused to the beta2 adrenergic receptor (beta2AR) and AC were incubated with BATP, the resultant mixture injected, and BATP separated from product BODIPY FL cAMP (BcAMP) by CE. AC activity was quantified by measuring the rate of BcAMP formation. beta2AR agonists isoproterenol and terbutaline increased basal AC activity with EC50s of 2.4 +/- 0.2 and 60 +/- 9 nM, respectively. The antagonist propranolol competed with terbutaline for beta2AR binding sites and expectedly right-shifted the terbutaline dose-response curve to 8 +/- 3 microM. The high sensitivity of the assay was demonstrated by detection of small changes in AC activity, with the partial agonist alprenolol increasing (22 +/- 1%) and the inverse agonist ICI 118,551 decreasing (19 +/- 2%) basal activity. The simplicity and automation of the CE-LIF assay offers advantages over the more traditional assay using radiochemical ATP and column chromatography.

Evaluation and comparison of very high pressure liquid chromatography systems for the separation and validation of pharmaceutical compounds.

Chromatography using sub-2 microm particles is becoming increasingly popular due to the potential for increased speed, resolution, sensitivity, and peak capacity. To meet the demand, various vendors have re-engineered traditional LC systems to operate at pressures of up to 15000 psi to accommodate the elevated backpressures associated with using sub-2 microm particles. This report investigates and compares the performance of three very high pressure LC (VHPLC) systems: Waters Acquity, Agilent 1200 SL, and Thermo Accela. Specifications for the pump, autosampler, column compartment, detector, and software for each instrument are presented. To assess the chromatographic performance of the three instruments, method development and validation were performed for three pharmaceutical compounds and the results are compared and discussed. The material presented herein serves to highlight the different features of the VHPLC instruments, and assess their suitability for the analysis of pharmaceutical compounds.

Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening.

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.

A CE assay for the detection of agonist-stimulated adenylyl cyclase activity.

A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein-coupled receptor (GPCR) level. In the assay, cell membranes overexpressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose-dependent manner. In both the CE-UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two- to three-fold maximum increase in AC activity with EC50s of 4.2 +/- 0.7 and 2.4 +/- 0.7 microM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the beta2-adrenergic receptor (beta2AR) fused to the stimulatory G protein. Terbutaline (beta2AR agonist) increased the basal rate of cAMP formation 1.7 +/- 0.1-fold resulting in an EC50 of 62 +/- 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC50 of terbutaline by the beta2AR antagonist propranolol. The CE-UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.

Detection of adenylyl cyclase activity using a fluorescent ATP substrate and capillary electrophoresis.

A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CE-LIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or Galpha(s)-GTPgammaS as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected EC50 values for forskolin and Galpha(s)-GTPgammaS of 27 +/- 6 microM and 317 +/- 56 nM, respectively. These EC50 values compared well to those previously reported using [alpha-32P]ATP as substrate. When AC was concurrently activated with 2.5 microM forskolin and 25 nM Galpha(s)-GTPgammaS, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2'-(or-3')-O-(N-methylanthraniloyl) guanosine 5'-triphosphate with an IC50 of 9 +/- 6 nM. The use of a fluorescent substrate combined with CE separation has enabled development of a rapid and robust method for detection of AC activity that is an attractive alternative to the AC assay using radioactive nucleotide and column chromatography. In addition, the assay has potential for high-throughput screening of drugs that act at AC.

Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection.

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.

Detection of G proteins by affinity probe capillary electrophoresis using a fluorescently labeled GTP analogue.

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.

Phospholipid bilayer coatings for the separation of proteins in capillary electrophoresis.

The double-chained, zwitterionic phospholipid 1,2-dilauroyl-sn-phosphatidylcholine (DLPC, C12) was investigated for its use as a wall coating for the prevention of protein adsorption in capillary electrophoresis. DLPC forms a semipermanent coating at the capillary wall, which allows excess phospholipid to be removed from the capillary prior to electrophoretic separation. A DLPC-coated capillary allowed for the separation of both cationic and anionic proteins with efficiencies as high as 1.4 million plates/m. Migration time reproducibility was less than 1.3% RSD from run to run and less than 4.0% RSD from day to day. Protein recovery was as high as 93%. Cationic and anionic proteins could be separated over a pH range of 3-10, all yielding good efficiencies (N up to 1 million plates/m). The chain length of the phospholipid affected the performance of the wall coating. The C10 analogue of DLPC (DDPC) did not form a coating on the capillary wall while the C14 analogue of DLPC (DMPC) formed a stable coating that prevented protein adsorption to the same extent as its C12 counterpart.