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Richard D Smith

Publications and source records attributed to Richard D Smith.

At least 19 recordsLinked to original sources
PubMed2026

Proteogenomic analysis of pediatric and AYA high-grade glioma reveals age-dependent biology, female-male differences, and kinase targets.

High-grade gliomas (HGGs) in children and adolescents and young adults (AYA) exhibit distinct biology across the neurodevelopmental spectrum. To dissect tumor-intrinsic molecular characteristics independent of developmental variation, we perform comprehensive proteogenomic analyses of tumors from 112 HGG patients aged 0-40 years. Our multi-omics analysis identifies two AYA subgroups-adolescents (aged 15-26 years) and young adults (aged 26-40 years)-with distinct molecular profiles and survival outcomes. Tumor-normal comparisons and survival modeling highlight roles of oxidative phosphorylation and neuronal system biology in glioma progression. Causal network analysis and cell line studies provide a rationale for personalized therapies targeting candidate kinases, such as CDK8. Survival modeling, clustering, and immune-landscape analyses identify proteins, post-translational modifications, and immune signatures linked to outcomes and reveal clinically relevant differences between male and female patients.

adolescent and young adult glioma↗
PubMed2025

Dysregulation of lung epithelial cell homeostasis and immunity contributes to Middle East respiratory syndrome coronavirus disease severity.

Coronaviruses (CoV) emerge suddenly from animal reservoirs to cause novel diseases in new hosts. Discovered in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in camels in the Middle East and is continually causing local outbreaks and epidemics. While all three newly emerging human CoVs from the past 20 years (SARS-CoV, SARS-CoV-2, and MERS-CoV) cause respiratory disease, each CoV has unique host interactions that drive differential pathogeneses. To better understand the virus and host interactions driving lethal MERS-CoV infection, we performed a longitudinal multi-omics analysis of sublethal and lethal MERS-CoV infection in mice. Significant differences were observed in body weight loss, virus titers, and acute lung injury among lethal and sub-lethal virus doses. Virus-induced apoptosis of type I and II alveolar epithelial cells suggests that loss or dysregulation of these key cell populations was a major driver of severe disease. Omics analysis suggested differential pathogenesis was multi-factorial with clear differences among innate and adaptive immune pathways as well as those that regulate lung epithelial homeostasis. Infection of mice lacking functional T and B cells showed that adaptive immunity was important in controlling viral replication but also increased pathogenesis. In summary, we provide a high-resolution host response atlas for MERS-CoV infection and disease severity. Multi-omics studies of viral pathogenesis offer a unique opportunity to not only better understand the molecular mechanisms of disease but also to identify genes and pathways that can be exploited for therapeutic intervention all of which is important for our future pandemic preparedness.IMPORTANCEEmerging coronaviruses like SARS-CoV, SARS-CoV-2, and MERS-CoV cause a range of disease outcomes in humans from an asymptomatic, moderate, and severe respiratory disease that can progress to death but the factors causing these disparate outcomes remain unclear. Understanding host responses to mild and life-threatening infections provides insight into virus-host networks within and across organ systems that contribute to disease outcomes. We used multi-omics approaches to comprehensively define the host response to moderate and severe MERS-CoV infection. Severe respiratory disease was associated with dysregulation of the immune response. Key lung epithelial cell populations that are essential for lung function get infected and die. Mice lacking key immune cell populations experienced greater virus replication but decreased disease severity implicating the immune system in both protective and pathogenic roles in response to MERS-CoV. These data could be utilized to design new therapeutic strategies targeting specific pathways that contribute to severe disease.

Animals↗
PubMed2013

The identification of novel potential injury mechanisms and candidate biomarkers in renal allograft rejection by quantitative proteomics.

Early transplant dysfunction and failure because of immunological and nonimmunological factors still presents a significant clinical problem for transplant recipients. A critical unmet need is the noninvasive detection and prediction of immune injury such that acute injury can be reversed by proactive immunosuppression titration. In this study, we used iTRAQ -based proteomic discovery and targeted ELISA validation to discover and validate candidate urine protein biomarkers from 262 renal allograft recipients with biopsy-confirmed allograft injury. Urine samples were randomly split into a training set of 108 patients and an independent validation set of 154 patients, which comprised the clinical biopsy-confirmed phenotypes of acute rejection (AR) (n = 74), stable graft (STA) (n = 74), chronic allograft injury (CAI) (n = 58), BK virus nephritis (BKVN) (n = 38), nephrotic syndrome (NS) (n = 8), and healthy, normal control (HC) (n = 10). A total of 389 proteins were measured that displayed differential abundances across urine specimens of the injury types (p < 0.05) with a significant finding that SUMO2 (small ubiquitin-related modifier 2) was identified as a "hub" protein for graft injury irrespective of causation. Sixty-nine urine proteins had differences in abundance (p < 0.01) in AR compared with stable graft, of which 12 proteins were up-regulated in AR with a mean fold increase of 2.8. Nine urine proteins were highly specific for AR because of their significant differences (p < 0.01; fold increase >1.5) from all other transplant categories (HLA class II protein HLA-DRB1, KRT14, HIST1H4B, FGG, ACTB, FGB, FGA, KRT7, DPP4). Increased levels of three of these proteins, fibrinogen beta (FGB; p = 0.04), fibrinogen gamma (FGG; p = 0.03), and HLA DRB1 (p = 0.003) were validated by ELISA in AR using an independent sample set. The fibrinogen proteins further segregated AR from BK virus nephritis (FGB p = 0.03, FGG p = 0.02), a finding that supports the utility of monitoring these urinary proteins for the specific and sensitive noninvasive diagnosis of acute renal allograft rejection.

Acute Kidney Injury↗
PubMed2004

Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS.

Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of approximately 10(3)) 15-microm-i.d. capillary liquid chromatography separations (i.e., using nanoLC, approximately 20 nL/min mobile-phase flow rate at the optimal linear velocity of approximately 0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., approximately 100 times larger than FTICR MS), corresponding to a sensitivity of approximately 7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.

Amino Acid Sequence↗
PubMed2004

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics.

We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dual-channel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (<20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ion-energy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10(3), and >10(5) for peptide abundances in the overall separation.

Amino Acid Sequence↗
PubMed2003

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures.

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.

Amino Acid Sequence↗
PubMed2003

Integration of electrokinetic-based multidimensional separation/concentration platform with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry for proteome analysis of Shewanella oneidensis.

This work focuses on the development of a multidimensional electrokinetic-based separation/concentration platform coupled with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) for achieving the high resolution and ultrasensitive analysis of complex protein/peptide mixtures. A microdialysis junction is employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) with transient capillary isotachophoresis/zone electrophoresis (CITP/CZE) in an integrated platform. Besides the excellent resolving power afforded by both CIEF and CZE separations, the electrokinetic focusing/stacking effects of CIEF and CITP greatly enhance the dynamic range and detection sensitivity of MS for protein identification. The constructed multidimensional separation/concentration platform is demonstrated for the analysis of Shewanella oneidensis proteome, which has considerable implications toward the bioremediation of environmental pollutants. The electrokinetic-based platform offers the overall peak capacity comparable to those obtained using multidimensional chromatography systems, but with a much shorter run time and no need for column regeneration. Most importantly, a total of 1174 unique proteins, corresponding to 26.5% proteome coverage, are identified from the cytosolic fraction of S. oneidensis, while requiring <500 ng of proteolytic digest loaded in the CIEF capillary. The ultrasensitive capabilities of electrokinetic-based proteome approach are attributed to the concentration effect in CIEF, the electrokinetic stacking of CITP, the nanoscale peak volume in CZE, the "accurate mass tag" strategy for protein/peptide identification, and the high-sensitivity, high-resolution, and high-mass measurement accuracy of FTICR-MS.

Amino Acid Sequence↗
PubMed2003

Duplex foldamers from assembly induced folding.

Oligoamide strands 1, 2, and 3, consisting of 4-H-bond units, were originally designed to form noncovalent polymers based on the expectation that they would adopt an extended conformation. Instead of assembling into the expected supramolecular polymer through their 4-H-bond units, the 1:1 mixture of 1 and 2 was found to form a highly stable dimeric species. To dimerize, the H-bonding sequences of 1 and 2 can only adopt a folded (stacked) conformation. The self-assembly of 3 was also found to adopt a similar folded duplex conformation. These novel duplex foldamers are very stable. They were characterized by 1D and 2D 1H NMR, VPO, and mass spectral (ESI) studies.

Biomimetic Materials↗
PubMed2003

Automated gain control and internal calibration with external ion accumulation capillary liquid chromatography-electrospray ionization Fourier transform ion cyclotron resonance.

When combined with capillary LC separations, electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) has demonstrated capabilities for advanced characterization of proteomes based upon analyses of proteolytic digests. Incorporation of external (to the ICR cell) multipole devices with FTICR for ion selection and ion accumulation has enhanced the dynamic range, sensitivity, and duty cycle of measurements. However, the highly variable ion production rate from an LC separation can result in "overfilling" of the external trap during the elution of major peaks and result in m/z discrimination and fragmentation of peptide ions. Excessive space charge trapped in the ICR cell also causes significant shifts in the detected ion cyclotron frequencies, reducing the achievable mass measurement accuracy (MMA) and making protein identification less effective. To eliminate m/z discrimination in the external ion trap, further increase duty cycle, and improve MMA, we have developed the capability for data-dependent adjustment of ion accumulation times in the course of an LC separation, referred to as automated gain control (AGC). This development has been implemented in combination with low kinetic energy gated ion trapping and internal calibration using a dual-channel electrodynamic ion funnel. The overall system was initially evaluated in the analysis of a tryptic digest of bovine serum albumin. In conjunction with internal calibration, the capillary LC-ESI-AGC-FTICR instrumentation provided a approximately 10-fold increase in the number of identified tryptic peptides compared to that obtained using a fixed ion accumulation time and external calibration methods.

Journal Article↗
PubMed2003

High-efficiency on-line solid-phase extraction coupling to 15-150-microm-i.d. column liquid chromatography for proteomic analysis.

The ability to manipulate and effectively utilize small proteomic samples is important for analyses using liquid chromatography (LC) in combination with mass spectrometry (MS) and becomes more challenging for very low flow rates due to extra column volume effects on separation quality. Here we report on the use of commercial switching valves (150-microm channels) for implementing the on-line coupling of capillary LC columns operated at 10,000 psi with relatively large solid-phase extraction (SPE) columns. With the use of optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained demonstrating peak capacities of approximately 1000 for capillaries having inner diameters between 15 and 150 microm. The on-line coupled SPE columns increased the sample processing capacity by approximately 400-fold for sample solution volume and approximately 10-fold for sample mass. The proteomic applications of this on-line SPE-capillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Using an ion trap tandem MS it was typically feasible to identify 1100-1500 unique peptides in a 5-h analysis. Peptides extracted from the SPE column and then eluted from the LC column covered a hydrophilicity/hydrophobicity range that included an estimated approximately 98% of all tryptic peptides. The SPE-capillary LC implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for automated proteomic analyses.

Amino Acid Sequence↗
PubMed2003

Use of artificial neural networks for the accurate prediction of peptide liquid chromatography elution times in proteome analyses.

The use of artificial neural networks (ANNs) is described for predicting the reversed-phase liquid chromatography retention times of peptides enzymatically digested from proteome-wide proteins. To enable the accurate comparison of the numerous LC/MS data sets, a genetic algorithm was developed to normalize the peptide retention data into a range (from 0 to 1), improving the peptide elution time reproducibility to approximately 1%. The network developed in this study was based on amino acid residue composition and consists of 20 input nodes, 2 hidden nodes, and 1 output node. A data set of approximately 7000 confidently identified peptides from the microorganism Deinococcus radiodurans was used for the training of the ANN. The ANN was then used to predict the elution times for another set of 5200 peptides tentatively identified by MS/MS from a different microorganism (Shewanella oneidensis). The model was found to predict the elution times of peptides with up to 54 amino acid residues (the longest peptide identified after tryptic digestion of S. oneidensis) with an average accuracy of approximately 3%. This predictive capability was then used to distinguish with high confidence isobar peptides otherwise indistinguishable by accurate mass measurements as well as to uncover peptide misidentifications. Thus, integration of ANN peptide elution time prediction in the proteomic research will increase both the number of protein identifications and their confidence.

Amino Acid Sequence↗
PubMed2003

High mass measurement accuracy determination for proteomics using multivariate regression fitting: application to electrospray ionization time-of-flight mass spectrometry.

Important factors that limit the mass measurement accuracy from a mass spectrometer are related to (1) the type of mass analyzer used and (2) the data processing/calibration methods used to obtain mass values from the raw data. Here, two data processing methods are presented that correct for systematic deviations when the mass of ions is measured using a time-of-flight (TOF) mass spectrometer. The first fitting method is one where m/z values are obtained from fitting peak distributions using double Gaussian functions. A second calibration method takes into account the slight nonlinear response of the TOF analyzer in addition to the drift in the calibration over time. Using multivariate regression, both of these two effects can be corrected for using a single calibration formula. Achievable performance was evaluated with a trypsin digestion of serum albumin and proteins from the organism D. radiodurans that was analyzed using gradient reversed-phase liquid chromatography combined with an electrospray ionization orthogonal TOF mass spectrometer. The root-mean-square deviation between the theoretical and experimental m/z values for serum albumin tryptic peptides was found to be 8 ppm using the double Gaussian-multivariate method compared to 29 ppm determined using linear calibration and normal peak centroiding. An advantage of the methods presented here is that no calibrant compounds need to be added to the mobile phase, thereby avoiding interference effects and signal suppression of analytes.

Bacterial Proteins↗
PubMed2003

Proteome analysis by mass spectrometry.

The coupling of high-performance mass spectrometry instrumentation with highly efficient chromatographic and electrophoretic separations has enabled rapid qualitative and quantitative analysis of thousands of proteins from minute samples of biological materials. Here, we review recent progress in the development and application of mass spectrometry-based techniques for the qualitative and quantitative analysis of global proteome samples derived from whole cells, tissues, or organisms. Techniques such as multidimensional peptide and protein separations coupled with mass spectrometry, accurate mass measurement of peptides from global proteome digests, and mass spectrometric characterization of intact proteins hold great promise for characterization of highly complex protein mixtures. Advances in chemical tagging and isotope labeling techniques have enabled quantitative analysis of proteomes, and highly specific isolation strategies have been developed aimed at selective analysis of posttranslationally modified proteins.

Chromatography, Affinity↗
PubMed2003

Construction of the contingent valuation market in health care: a critical assessment.

Contingent valuation (CV) has been criticised for being too hypothetical, with expressed values bearing little relation to actual values. The magnitude of this divergence, however, depends upon how realistic and believable the contingent market is. This paper presents an overview of five key aspects in the construction of the contingent market: (i) scenario development and presentation; (ii) payment vehicle; (iii) expression of risk; (iv) time period of valuation; and (v) survey administration. CV studies in health care since 1985, totalling 111, are critically reviewed with respect to these five aspects. It is concluded that CV studies in health care have performed poorly in the construction, specification and presentation of the contingent market, and that there has been little, if any, improvement in this respect over the last 15 years. Suggestions are made concerning why this may be the case, and how the construction of the contingent market may be improved in future.

Attitude to Health↗