EMSL: Dick Smith's Publications

Alquier T, ML Peyot, MG Latour, M Kebede, CM Sorensen, S Gesta, CR Kahn, RD Smith, TL Jetton, TO Metz, M Prentki, and VJ Poitout. 2009. "Deletion of GPR40 Impairs Glucose-Induced Insulin Secretion In Vivo in Mice Without Affecting Intracellular Fuel Metabolism in Islets ." Diabetes 58(11):2607-2615. Abstract The G protein-coupled receptor GPR40 mediates fatty-acid potentiation of glucose-stimulated insulin secretion, but its contribution to insulin secretion in vivo and mechanisms of action remain uncertain. This study was aimed to ascertain whether GPR40 controls insulin secretion in vivo and modulates intracellular fuel metabolism in islets. We observed that glucose- and arginine-stimulated insulin secretion, assessed by hyperglycemic clamps, was decreased by approximately 60% in GPR40 knock-out (KO) fasted and fed mice, without changes in insulin sensitivity assessed by hyperinsulinemic-euglycemic clamps. Glucose and palmitate metabolism were not affected by GPR40 deletion. Lipid profiling revealed a similar increase in triglyceride and decrease in lysophosphatidylethanolamine species in WT and KO islets in response to palmitate. These results demonstrate that GPR40 regulates insulin secretion in vivo not only in response to fatty acids but also to glucose and arginine, without altering intracellular fuel metabolism.

Ansong C, H Yoon, S Porwollik, H Mottaz-Brewer, BO Petritis, N Jaitly, JN Adkins, M Mcclelland, F Heffron, and RD Smith. 2009. "Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation." PLoS One 4(3):e4809. doi:10.1371/journal.pone.0004809 Abstract In recent years the profound importance of sRNA-mediated translational/post-transcriptional regulation has been increasingly appreciated. However, the global role played by translational regulation in control of gene expression has never been elucidated in any organism for the simple reason that global proteomics methods required to accurately characterize post-transcriptional processes and the knowledge of translational control mechanisms have only become available within the last few years. The proteins Hfq and SmpB are essential for the biological activity of a range of regulatory sRNAs and thus provide a means to identify potential targets of sRNA regulation. We performed a sample-matched global proteomics and transcriptional analysis to examine the role of Hfq and SmpB in global protein translation and virulence using the Salmonella typhimurium model system. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all Salmonella proteins, respectively, with limited correlation between transcription and protein expression. This is the first report suggesting that SmpB could be a general translational regulator. The broad spectrum of proteins modulated by Hfq was also surprising including central metabolism, LPS biosynthesis, two-component regulatory systems, quorum sensing, SP1-TTSS, oxidative stress, fatty acid metabolism, nucleoside and nucleotide metabolism, envelope stress, aminoacyl-tRNA synthetases, amino acid biosynthesis, peptide transport, and motility.. The extent of global regulation of translation by Hfq is unexpected, with profound effects in all stages of Salmonella’s life cycle. Our results represent the first global systems-level analysis of translational regulation; the elucidated potential targets of sRNA regulation from our analysis will facilitate the characterization of sRNA control mechanisms and their targets mRNAs by the broader scientific community by providing global-scale insights.

Chowdhury SM, L Shi, H Yoon, C Ansong, LM Rommereim, AD Norbeck, KJ Auberry, RJ Moore, JN Adkins, F Heffron, and RD Smith. 2009. "A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis ." Journal of Proteome Research 8(3):1504-1514 . Abstract We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins― HimD, PduB and PhoP― with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .

Heibeck TH, SJ Ding, LK Opresko, R Zhao, AA Schepmoes, F Yang, AV Tolmachev, ME Monroe, DG Camp, II, RD Smith, HS Wiley, and W Qian. 2009. "An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells." Journal of Proteome Research 8(8):3852-3861. Abstract Protein tyrosine phosphorylation is a central regulatory mechanism in cell signaling. To extensively characterize the site-specific tyrosine phosphorylation in human cells, we present here a global survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying anti-phosphotyrosine (pTyr) peptide immunoaffinity purification (IP) coupled with high sensitivity LC-MS/MS. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and an acute stimulated condition with epidermal growth factor (EGF). The estimated false discovery rate is 1.0% as measured by comparison against a scrambled database search. Comparison of these data to the literature showed significant agreement in site matches. Additionally 281 sites were not previously observed in HMEC culture were found. Twenty-nine of these sites have not been reported in any human cell or tissue system. The global profiling also allowed us to examine the phosphorylation stoichiometry differences based on spectral count information. Comparison of the data to a previous global proteome profiling study illustrates that most of the highly phoshorylated proteins are of relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed for many of the identified proteins, suggesting potentially more important functional roles for those highly phosphorylated pTyr sites within a given protein. By mapping to major signaling networks such as EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which should allow us to select interesting targeted involved in a given pathway for more directed studies. This extensive HMEC tyrosine phosphorylation dataset represents an important database resource for facilitating future hypothesis-driven or targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems.

Jaitly N, AM Mayampurath, KA Littlefield, JN Adkins, GA Anderson, and RD Smith. 2009. "Decon2LS: An Open-Source Software Package for Automated Processing and Visualization of High Resolution Mass Spectrometry Data." BMC Bioinformatics 10:, Abstract Data generated from liquid chromatography coupled to high-resolution mass spectrometry (LC-MS)-based studies of a biological sample can contain large amounts of biologically significant information in the form of proteins, peptides, and metabolites. Interpreting these data uses the mass spectra to infer masses and abundances of biomolecules injected into the instrument. Because of the inherent complexity of mass spectral patterns produced by these biomolecules, the analysis is significantly enhanced by using visualization capabilities to inspect and confirm results. In this paper we describe Decon2LS, an open-source software package for automated processing and visualization of high-resolution MS data. Drawing extensively on algorithms developed over the last ten years for ICR2LS, Decon2LS packages the algorithms as a rich set of modular, reusable processing classes for performing diverse functions such as raw data readers, routine peak finding, theoretical isotope distribution modelling, and deisotoping. Because the source code is openly available, these functionalities can now be used to build derivative applications in relatively fast manner. In addition, Decon2LS provides an extensive set of visualization tools, such as high performance chart controls.

Karpievitch Y, JR Stanley, T Taverner, J Huang, JN Adkins, C Ansong, F Heffron, TO Metz, W Qian, H Yoon, RD Smith, and AR Dabney. 2009. "AA statistical framework for protein quantitation in bottom-up MS-based proteomics ." Bioinformatics 25(16):2028-2034. doi:10.1093/bioinformatics/btp362 Abstract ABSTRACT Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and confidence measures. Challenges include the presence of low-quality or incorrectly identified peptides and widespread, informative, missing data. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model for protein abundance in terms of peptide peak intensities, applicable to both label-based and label-free quantitation experiments. The model allows for both random and censoring missingness mechanisms and provides naturally for protein-level estimates and confidence measures. The model is also used to derive automated filtering and imputation routines. Three LC-MS datasets are used to illustrate the methods. Availability: The software has been made available in the open-source proteomics platform DAnTE (Polpitiya et al. (2008)) (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu

Kelly RT, JS Page, I Marginean, K Tang, and RD Smith. 2009. "Dilution-Free Analysis from Picoliter Droplets by Nano-Electrospray Ionization Mass Spectrometry." Angewandte Chemie International Edition 48(37):6832-6835. doi:10.1002/anie.200902501 Abstract The expanding role of microfluidics for chemical and biochemical analysis is due to factors including the favorable scaling of separation performance with reduced channel dimensions,[1] flexibility afforded by computer-aided device design, and the ability to integrate multiple sample handling and analysis steps into a single platform.[2] Such devices enable smaller liquid volumes and sample sizes to be handled than can be achieved on the benchtop, where sub-microliter volumes are difficult to work with and where sample losses to the surfaces of multiple reaction vessels become prohibitive. A particularly attractive microfluidic platform for sample-limited analyses employs aqueous droplets or plugs encapsulated by an immiscible oil.[3,4] Each droplet serves as a discrete compartment or reaction chamber enabling, e.g., high throughput screening[5,6] and kinetic studies[7-9] of femto- to nanoliter samples, as well as the encapsulation[10-12] and lysis[10] of individual cells with limited dilution of the cellular contents

Kim BC, D Lopez-Ferrer, S Lee, H Ahn, S Nair, SH Kim, BS Kim, K Petritis, DG Camp, II, JW Grate, RD Smith, Y Koo, MB Gu, and J Kim. 2009. "Highly Stable Trypsin-Aggregate Coatings on Polymer Nanofibers for Repeated Protein Digestion." Proteomics 9(7):1893-1900. Abstract A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This new process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was also resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with enzyme aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

Lopez-Ferrer D, KK Hixson, HS Smallwood, TC Squier, K Petritis, and RD Smith. 2009. "Evaluation of a High Intensity Focused Ultrasound-Immobilized Trypsin Digestion and 18 O-Labeling Method for Quantitative Proteomics." Analytical Chemistry 81(15):6272-6277. Abstract A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultra-rapid digestion and thorough 18O labeling for quantitative protein comparisons. The reproducible and highly efficient method provided effective digestions in <1 min and minimized the amount of enzyme required compared to traditional methods. This method was demonstrated for digestion of both simple and complex protein mixtures, including bovine serum albumin, a global proteome extract from bacteria Shewanella oneidensis, and mouse plasma, as well as for the labeling of complex protein mixtures, which validated the application of this method for differential proteomic measurements. This approach is simple, reproducible, cost effective, and rapid, and thus well-suited for automation.

Mabrouki RB, RT Kelly, DC Prior, AA Shvartsburg, K Tang, and RD Smith. 2009. "Improving FAIMS Sensitivity using a Planar Geometry with Slit Interfaces." Journal of the American Society for Mass Spectrometry 20(9):1768-1774. doi:10.1016/j.jasms.2009.05.019 Abstract Differential mobility spectrometry or field asymmetric waveform ion mobility spectrometry (FAIMS) is gaining broad acceptance for analyses of gas-phase ions, especially in conjunction with largely orthogonal separation methods such as mass spectrometry (MS) and/or conventional (drift tube) ion mobility spectrometry. In FAIMS, ions are filtered while passing through a gap between two electrodes that may have planar or curved (in particular, cylindrical) geometry. Despite substantial inherent advantages of the planar configuration and its universal acceptance in stand-alone FAIMS devices, commercial FAIMS/MS systems have employed curved FAIMS geometries that could be interfaced to MS more effectively. Here we report a new planar (p-) FAIMS design with slit-shaped entrance and exit apertures that substantially increase ion transmission in and out of the analyzer. The front slit interface effectively couples p-FAIMS to multi-emitter electrospray ionization (ESI) sources, improving greatly the ion current introduced to the device. The back slit interface increases the transmission of ribbon-shaped ion beams output by the p-FAIMS to downstream stages such as a MS. Overall, the ion signal in ESI/FAIMS/MS analyses is raised by over an order of magnitude without affecting the FAIMS resolution.