Proteomic identification of synaptic caspase substrates.

The dismantling and elimination of excess neurons and their connections (pruning) is essential for brain development and may be aberrantly reactivated in some neurodegenerative diseases. Growing evidence implicates caspase-mediated apoptotic and nonapoptotic cascades in the dysfunction and death of neurons in neurodegenerative disorders such as Alzheimer's, Parkinson, and Huntington's diseases. It is the cleaved caspase substrates that are the effectors of synapse elimination. However, their identities, specific cleavage sites, and functional consequences of cleavage are largely unknown. An important gap in our knowledge is a comprehensive catalog of synapse-specific or synapse-enriched caspase targets. Traditional biochemical approaches have revealed only a small number of neuronal caspase targets. Instead, we utilized a gel-based proteomics approach to enable the first global analysis of caspase-mediated cleavage events in mammalian brain synapses, employing both an in vitro system with recombinant activated caspases and an in vivo model of ethanol-induced neuronal apoptosis. Of the more than 70 putative cleavage substrates that were identified, 22 were previously known caspase substrates. Among the novel targets identified and validated by Western blot were the proton pump ATPase subunit ATP6V1B2 and the N-ethylmaleimide-sensitive fusion protein (NSF). Our work represents the first comprehensive, proteome-wide screen for proteolytic targets of caspases in neuronal synapses. Our discoveries will have significance for both furthering basic understanding of roles of caspases in synaptic plasticity and synaptic loss in neurodegeneration, and on a more immediately practical level, may provide candidate biomarkers for measuring synapse loss in human disease states.

Method Development for Container Closure Integrity Evaluation via Headspace Gas Ingress by Using Frequency Modulation Spectroscopy.

USP <1207.1> Section 3.5 states that "A deterministic leak test method having the ability to detect leaks at the product's maximum allowable leakage limit is preferred when establishing the inherent integrity of a container-closure system." Ideally, container closure integrity of parenteral packaging would be evaluated by measuring a physical property that is sensitive to the presence of any package defect that breaches package integrity by increasing its leakage above its maximum allowable leakage limit. The primary goals of the work presented herein were to demonstrate the viability of the nondestructive, deterministic method known as laser-based gas headspace analysis for evaluating container closure integrity and to provide a physical model for predicting leak rates for a variety of container volumes, headspace conditions, and defect sizes. The results demonstrate that laser-based headspace analysis provides sensitive, accurate, and reproducible measurements of the gas ingress into glass vial-stopper package assemblies that are under either diffusive or effusive leak conditions. Two different types of positive controls were examined. First, laser-drilled micro-holes in thin metal disks that were crimped on top of 15R glass vials served as positive controls with a well-characterized defect geometry. For these, a strong correlation was observed between the measured ingress parameter and the size of the defect for both diffusive and effusive conditions. Second, laser-drilled holes in the wall of glass vials served as controls that more closely simulate real-world defects. Due to their complex defect geometries, their diffusive and effusive ingress parameters did not necessarily correlate; this is an important observation that has significant implications for standardizing the characterization of container defects. Regardless, laser-based headspace analysis could readily differentiate positive and negative controls for all leak conditions, and the results provide a guide for method development of container closure integrity tests.LAY ABSTRACT: The new USP 39 <1207>, "Package Integrity Evaluation-Sterile Products", states in section 3.4.1: "tracer gas tests performed using … laser-based gas headspace analysis [have] been shown to be sensitive enough to quantitatively analyze leakage through the smallest leak paths found to pose the smallest chance of liquid leakage or microbial ingress in rigid packaging." In addition, USP <1207> also states that "for such methods, the limit of detection can be mathematically predicted on the basis of gas flow kinetics." Using the above statements as a foundation, this paper presents a theoretical basis for predicting the gas ingress through well-defined defects in product vials sealed under a variety of headspace conditions. These calculated predictions were experimentally validated by comparing them to measurements of changes in the headspace oxygen content or total pressure for several different positive controls using laser-based headspace analysis. The results demonstrated that laser-based headspace analysis can, by readily differentiating between negative controls and positive controls with a range of defect sizes on the micron scale, be used to assess container closure integrity. The work also demontrated that caution must be used when attempting to correlate a leak rate to an idealized defect-size parameter.

Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells.

OBJECTIVES: Exosomes are 50-90nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells. DESIGN AND METHODS: For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines. RESULTS: Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multivesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells. CONCLUSION: Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system.

Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.

The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.

Translational dynamics of water at the phospholipid interface.

The residual water-proton magnetic relaxation dispersion profile obtained from suspensions of phospholipid vesicles in deuterium oxide was found to be a logarithmic function of the proton Larmor frequency at high magnetic field strengths, and independent of Larmor frequency at low magnetic field strengths. The residual proton relaxation is caused by dipole-dipole coupling between the residual water proton in otherwise deuterated water and the phospholipid protons. The logarithmic dependence on magnetic field strength is the signature of water-proton diffusive exploration on the interface that is approximately two-dimensionally constrained. Application of relaxation theory for two-dimensional diffusion to the spin-lattice relaxation data yields a translational correlation time of approximately 70 ps for water diffusing in the interface of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles.

Proteomic profile of reversible protein oxidation using PROP, purification of reversibly oxidized proteins.

Signal transduction pathways that are modulated by thiol oxidation events are beginning to be uncovered, but these discoveries are limited by the availability of relatively few analytical methods to examine protein oxidation compared to other signaling events such as protein phosphorylation. We report here the coupling of PROP, a method to purify reversibly oxidized proteins, with the proteomic identification of the purified mixture using mass spectrometry. A gene ontology (GO), KEGG enrichment and Wikipathways analysis of the identified proteins indicated a significant enrichment in proteins associated with both translation and mRNA splicing. This methodology also enabled the identification of some of the specific cysteine residue targets within identified proteins that are reversibly oxidized by hydrogen peroxide treatment of intact cells. From these identifications, we determined a potential consensus sequence motif associated with oxidized cysteine residues. Furthermore, because we identified proteins and specific sites of oxidation from both abundant proteins and from far less abundant signaling proteins (e.g. hepatoma derived growth factor, prostaglandin E synthase 3), the results suggest that the PROP procedure was efficient. Thus, this PROP-proteomics methodology offers a sensitive means to identify biologically relevant redox signaling events that occur within intact cells.

PICquant: a quantitative platform to measure differential peptide abundance using dual-isotopic labeling with 12C6- and 13C6-phenyl isocyanate.

We have developed a complete system for the isotopic labeling, fractionation, and automated quantification of differentially expressed peptides that significantly facilitates candidate biomarker discovery. We describe a new stable mass tagging reagent pair, (12)C(6)- and (13)C(6)-phenyl isocyanate (PIC), that offers significant advantages over currently available tags. Peptides are labeled predominantly at their amino termini and exhibit elution profiles that are independent of label isotope. Importantly, PIC-labeled peptides have unique neutral-mass losses upon CID fragmentation that enable charge state and label isotope identification and, thereby, decouple the sequence identification from the quantification of candidate biomarkers. To exploit these properties, we have coupled peptide fractionation protocols with a Thermo LTQ-XL LC-MS(2) data acquisition strategy and a suite of automated spectrum analysis software that identifies quantitative differences between labeled samples. This approach, dubbed the PICquant platform, is independent of protein sequence identification and excludes unlabeled peptides that otherwise confound biomarker discovery. Application of the PICquant platform to a set of complex clinical samples showed that the system allows rapid identification of peptides that are differentially expressed between control and patient groups.

MAZIE: a mass and charge inference engine to enhance database searching of tandem mass spectra.

Peptide sequence identification using tandem mass spectroscopy remains a major challenge for complex proteomic studies. Peptide matching algorithms require the accurate determination of both the mass and charge of the precursor ion and accommodate uncertainties in these properties by using a wide precursor mass tolerance and by testing, for each spectrum, several possible candidate charges. Using a data acquisition strategy that includes obtaining narrow mass-range MS(1) "zoom" scans, we describe here a post-acquisition algorithm dubbed mass and charge (Z) inference engine (MAZIE), which accurately determines the charge and monoisotopic mass of precursor ions on a low-resolution Thermo LTQ-XL mass spectrometer. This is achieved by examining the isotopic distribution obtained in the preceding MS(1) zoom spectrum and comparing to theoretical distributions for candidate charge states from +1 to +4. MAZIE then writes modified data files with the corrected monoisotopic mass and charge. We have validated MAZIE results by comparing the sequence search results obtained with the MAZIE-generated data files to results using the unmodified data files. Using two different search algorithms and a false discovery rate filter, we found that MAZIE-interpreted data resulted in 80% (using SEQUEST) and 30% (using OMSSA) more high-confidence sequence identifications. Analyses of these results indicate that the accurate determination of the precursor ion mass greatly facilitates the ability to differentiate between true and false positive matches, while the determination of the precursor ion charge reduces the overall search time but does not significantly reduce the ambiguity of interpreting the search results. MAZIE is distributed as an open-source PERL script.

Magnetic relaxation dispersion of lithium ion in solutions of DNA.

The magnetic field dependence of the nuclear spin-lattice relaxation rate constant defines the magnetic relaxation dispersion (MRD) and provides a direct characterization of the molecular dynamics that cause fluctuations in the magnetic couplings in the system and may also indicate the dimensional constraints on the motion. The counterion cloud surrounding a linear polyelectrolyte ion, such as DNA in solution, provides an interesting opportunity for ion confinement that helps in understanding the thermodynamics and the dynamics of the interactions between the polyion and other solutes. The MRD profiles of lithium ion and tetramethylammonium ion were recorded in dilute aqueous solutions of native calf thymus DNA, which provides a long, charged rod that reorients slowly. The 7Li ion relaxes through the nuclear electric quadrupole coupling and the proton-lithium dipole-dipole coupling; the protons of the tetramethylammonium ion relax by dipole-dipole coupling. MRD profiles of the 7Li+ ion are dominated by transient interactions with the DNA that yield a linear dependence of the spin-lattice relaxation rate constant on the logarithm of the Larmor frequency. This magnetic field dependence is consistent with diffusive ion motions that modulate two spatial coordinates that characterize the relaxation couplings in the vicinity of the polyion. Motions around the rod and fluctuations in the ion distance from the rod are consistent with these constraints for lithium. The magnetic field dependence of the tetramethylammonium ion proton relaxation rate constant is weak, but also approximately a linear function of the logarithm of the Larmor frequency, which implies that the field dependence is caused in part by local order in the DNA solution.