Ronald Moore - Profile on Academia.edu (original) (raw)

Papers by Ronald Moore

J Proteome Res, 2010

A burn injury represents one of the most severe forms of human trauma and is responsible for sign... more A burn injury represents one of the most severe forms of human trauma and is responsible for significant mortality worldwide. Here, we present the first quantitative proteomics investigation of the blood plasma proteome response to severe burn injury by comparing the plasma protein concentrations of 10 healthy control subjects with those of 15 severe burn patients at two time-points following the injury. The overall analytical strategy for this work integrated immunoaffinity depletion of the 12 most abundant plasma proteins with cysteinyl-peptide enrichment-based fractionation prior to LC-MS analyses of individual patient samples. Incorporation of an 18 O-labeled "universal" reference among the sample sets enabled precise relative quantification across samples. In total, 313 plasma proteins confidently identified with two or more unique peptides were quantified. Following statistical analysis, 110 proteins exhibited significant abundance changes in response to the burn injury. The observed changes in protein concentrations suggest significant inflammatory and hypermetabolic response to the injury, which is supported by the fact that many of the identified proteins are associated with acute phase response signaling, the complement system, and coagulation system pathways. The regulation of ∼35 proteins observed in this study is in agreement with previous results reported for inflammatory or burn response, but approximately 50 potentially novel proteins previously not known to be associated with burn response or inflammation are also found. Elucidating proteins involved in the response to severe burn injury may reveal novel targets for therapeutic interventions as well as potential predictive biomarkers for patient outcomes such as multiple organ failure.

Analytical Chemistry, 2005

The throughput of proteomics measurements that provide broad protein coverage is limited by the q... more The throughput of proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations as well as the subsequent mass spectrometric analysis; at present, analysis times can range anywhere from hours (high throughput) to days or longer (low throughput). We have explored the basis for proteomics analyses conducted on the order of minutes using high-speed capillary RPLC combined through online electrospray ionization interface with high-accuracy mass spectrometry (MS) measurements. Short 0.8-µm porous C18 particle-packed 50-µm-i.d. capillaries were used to speed the RPLC separations while still providing high-quality separations. Both time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS were applied for identifying peptides using the accurate mass and time (AMT) tag approach. Peptide RPLC relative retention (elution) times that were generated by solvent gradients that differed by at least 25-fold were found to provide relative elution times that agreed to within 5%, which provides the basis for using peptide AMT tags for higher throughput proteomics measurements. For fast MS acquisition speeds (e.g., 0.2 s for TOF and either ∼0.3 or ∼0.6 s for FTICR), peptide mass measurement accuracies of better than (15 ppm were obtained with the highspeed RPLC separations. The ability to identify peptides and the overall proteome coverage was determined by factors that include the separation peak capacity, the sensitivity of the MS (with fast scanning), and the accuracy of both the mass measurements and the relative RPLC peptide elution times. The experimental RPLC relative elution time accuracies of 5% (using high-speed capillary RPLC) and mass measurement accuracies of better than (15 ppm allowed for the confident identification of >2800 peptides and >760 proteins from >13 000 dif-ferent putative peptides detected from a Shewanella oneidensis tryptic digest. Initial results for both RPLC-ESI-TOF and RPLC-ESI-FTICR MS were similar, with ∼2000 different peptides from ∼600 different proteins identified within 2-3 min. For <120-s proteomic analysis, TOF MS analyses were more effective, while FTICR MS was more effective for the >150-s analysis due to the improved mass accuracies attained using longer spectrum acquisition times.

Global Analysis of Deinococcus Radiodurans Proteome by Csing Accurate Mass Tags

Pnas, 2002

Understanding biological systems and the roles of their constituents is facilitated by the abilit... more Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organism&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry

The Analyst, Jan 6, 2016

Understanding how biological molecules are generated, metabolized and eliminated in living system... more Understanding how biological molecules are generated, metabolized and eliminated in living systems is important for interpreting processes such as immune response and disease pathology. While genomic and proteomic studies have provided vast amounts of information over the last several decades, interest in lipidomics has also grown due to improved analytical technologies revealing altered lipid metabolism in type 2 diabetes, cancer, and lipid storage disease. Mass spectrometry (MS) measurements are currently the dominant approach for characterizing the lipidome by providing detailed information on the spatial and temporal composition of lipids. However, interpreting lipids' biological roles is challenging due to the existence of numerous structural and stereoisomers (i.e. distinct acyl chain and double-bond positions), which are often unresolvable using present approaches. Here we show that combining liquid chromatography (LC) and structurally-based ion mobility spectrometry (IMS...

SNaPP: Simplified Nano-Proteomics Platform for reproducible global proteomic analysis of nanogram protein quantities

Endocrinology, Jan 8, 2016

Global proteomic analyses of complex protein samples in nanogram quantities require a fastidious ... more Global proteomic analyses of complex protein samples in nanogram quantities require a fastidious approach to achieve in-depth protein coverage and quantitative reproducibility. Biological samples are often severely mass-limited and can preclude the application of more robust bulk sample processing workflows. In this study, we present a system that minimizes sample handling by using online immobilized trypsin digestion and solid phase extraction to create a simple, sensitive, robust, and reproducible platform for the analysis of nanogram-size proteomic samples. To demonstrate the effectiveness of our Simplified Nano-Proteomics Platform (SNaPP), we utilized the system to analyze preimplantation blastocysts collected on day 4 of pregnancy by flushing the uterine horns with saline. For each of our 3 sample groups, blastocysts were pooled from 3 mice resulting in 22, 22, and 25 blastocysts, respectively. The resulting proteomic data provides novel insight into mouse blastocyst protein ex...

Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

J Proteome Res, 2005

Most current methods for purification and identification of protein complexes use endogenous expr... more Most current methods for purification and identification of protein complexes use endogenous expression of affinity-tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, and gel separation and in-gel digestion prior to mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pull-down assay with denaturing elution, trypsin digestion in organic solvent, and LC-ESI MS/MS protein identification using SEQUEST analysis. Our method is simple and easy to scale-up and automate, making it suitable for high-throughput mapping of protein interaction networks and functional proteomics.

Applied and Environmental Microbiology, Nov 30, 2012

Cultures of the cyanobacterial genus Cyanothece have been shown to produce high 21 levels of bioh... more Cultures of the cyanobacterial genus Cyanothece have been shown to produce high 21 levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal 22 cycles when grown under N 2 -fixing conditions in light-dark cycles. We seek to better 23 understand the way in which proteins respond to these diurnal changes and we 24 performed quantitative proteome analysis of Cyanothece ATCC 51142 and PCC 7822 25 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N 2 -26 fixing conditions, and in the absence of glycerol, nitrogenase gene expression was 27 linked to the dark period. However, glycerol induced expression of nitrogenase during 28 part of the light period, together with cytochrome c oxidase (Cox), glycogen 29 phosphorylase (Glp), and glycolytic and pentose-phosphate pathway (PPP) enzymes. 30 This indicated that nitrogenase expression in the light was facilitated via higher 31 respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited in 32 Cyanothece ATCC 51142 in the presence of glycerol under H 2 producing conditions, 33 suggesting a competition between these sources of carbon. However, in Cyanothece 34 PCC 7822, the Calvin cycle still played a role in cofactor recycling during H 2 production. 35 Our data comprise the first comprehensive profiling of proteome changes in Cyanothece 36 PCC 7822, and allows an in-depth comparative analysis of major physiological and 37 biochemical processes that influence H 2 -production in both strains. Our results 38 revealed many previously uncharacterized proteins that may play a role in nitrogenase 39 activity and in other metabolic pathways and may provide suitable targets for genetic 40 manipulation that would lead to improvement of large scale H 2 production.

Journal of Proteome Research, Aug 1, 2008

A new method for rapid proteolytic digestion of proteins under high pressure that uses pressure c... more A new method for rapid proteolytic digestion of proteins under high pressure that uses pressure cycling technology in the range of 5-35 kpsi was demonstrated for proteomic analysis. Successful in-solution digestions of single proteins and complex protein mixtures were achieved in 60 s and then analyzed by reversed phase liquid chromatography-electrospray ionization ion trap-mass spectrometry. Method performance in terms of the number of Shewanella oneidensis peptides and proteins identified in a shotgun approach was evaluated relative to a traditional "overnight" sample preparation method. Advantages of the new method include greatly simplified sample processing, easy implementation, no cross contamination among samples, and cost effectiveness.

Method and Apparatus for Continuous Fluid Leak Monitoring and Detection in Analytical Instruments and Instrument Systems

ABSTRACT A method and device are disclosed that provide for detection of fluid leaks in analytica... more ABSTRACT A method and device are disclosed that provide for detection of fluid leaks in analytical instruments and instrument systems. The leak detection device includes a collection tube, a fluid absorbing material, and a circuit that electrically couples to an indicator device. When assembled, the leak detection device detects and monitors for fluid leaks, providing a preselected response in conjunction with the indicator device when contacted by a fluid.

Analytical Chemistry, Jun 1, 2005

Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; how... more Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; however, variations providing cutting-edge RPLC performance have generally not been adopted even though their benefits are well established. Here, we describe an automated format 20 kpsi RPLC system for proteomics and metabolomics that includes on-line coupling of micro-solid phase extraction for sample loading and allows electrospray ionization emitters to be readily replaced. The system uses 50 μm i.d. × 40-200 cm fused-silica capillaries packed with 1.4-3-μm porous C18-bonded silica particles to obtain chromatographic peak capacities of 1000-1500 for complex peptide and metabolite mixtures. This separation quality provided high-confidence identifications of >12 000 different tryptic peptides from >2000 distinct Shewanella oneidensis proteins (∼40% of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The protein identification reproducibility approached 90% between replicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207 proteins were identified in 120 min through assignment of 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS was approximately 10 6 based on analyses of a human blood plasma sample, for which 835 distinct proteins were identified with high confidence in a single 12-h run. A single run of the 20 kpsi RPLC-accurate mass MS detected >5000 different compounds from a metabolomics sample.

Preparation of 20-�m-i.d. Silica-based Monolithic Columns and Application for Proteomic Analysis

Blood serum is a complex body fluid that contains various proteins ranging in concentration over ... more Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.

NIH Public Access

PROTEOMICS

Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants

PLOS ONE, 2015

Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive ... more Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.

An Orthogonal Injection Ion Funnel Interface Providing Enhanced Performance for Selected Reaction Monitoring-Triple Quadruple Mass Spectrometry

Analytical chemistry, Jan 24, 2015

The electrodynamic ion funnel facilitates efficient focusing and transfer of charged particles in... more The electrodynamic ion funnel facilitates efficient focusing and transfer of charged particles in the higher pressure regions (e.g. ion source interfaces) of mass spectrometers, and thus provides increased sensitivity. An "off-axis" ion funnel design has been developed to reduce the source contamination and interferences from, e.g. ESI droplet residue and other poorly focused neutral or charged particles with very high mass-to charge ratios. In this study a dual ion funnel interface consisting of an orthogonal higher pressure electrodynamic ion funnel (HPIF) and an ion funnel trap combined with a triple quadrupole mass spectrometer was developed and characterized. An orthogonal ion injection inlet and a repeller plate electrode was used to direct ions to an ion funnel HPIF at 9-10 Torr pressure. Key factors for the HPIF performance characterized included the effects of RF amplitude, the DC gradient, and operating pressure. Compared to the triple quadrupole standard interfa...

Enhancing bottom-up and top-down proteomic measurements with ion mobility separations

Proteomics, Jan 5, 2015

Proteomic measurements with greater throughput, sensitivity and structural information are essent... more Proteomic measurements with greater throughput, sensitivity and structural information are essential for improving both in-depth characterization of complex mixtures and targeted studies. While liquid chromatography separation coupled with mass spectrometry (LC-MS) measurements have provided information on thousands of proteins in different sample types, the introduction of a separation stage that provides further component resolution and rapid structural information has many benefits in proteomic analyses. Technical advances in ion transmission and data acquisition have made ion mobility separations an opportune technology to be easily and effectively incorporated into LC-MS proteomic measurements for enhancing their information content. Herein, we report on applications illustrating increased sensitivity, throughput, and structural information by utilizing IMS-MS and LC-IMS-MS measurements for both bottom-up and top-down proteomics measurements. This article is protected by copyri...

Membrane Proteome Quantification Using Membrane-Impermeable Chemical Probe and Stable Isotopic Labeling

In this study, we have developed a large scale membrane proteome quantification strategy by combi... more In this study, we have developed a large scale membrane proteome quantification strategy by combining membrane protein enrichment using a membrane-impermeable chemical probe and quantifications using high-performance mass spectrometry and stable isotopic labeling. Membrane-impermeable chemical probe has been applied for the enrichment of membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and environmental electron receptors. LC/MS/MS analysis resulted in identifying 2,780 unique peptides from 321 membrane proteins which corresponded to 87% and 79% of all the identified unique peptides and proteins in the experiment, respectively. To demonstrate that the proposed strategy was specific and sensitive to protein abundance change in cell membranes, quantifications of enriched membrane proteins by stable isotopic labeling were further carried out for ...