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Recent Advances in Droplet-Based Microfluidic Technologies
micromachines Review Recent Advances in Droplet-based Microfluidic Technologies for Biochemistry and Molecular Biology 1, 1, 2, Joel Sánchez Barea y , Juhwa Lee y and Dong-Ku Kang * 1 Department of Chemistry, Incheon National University, Incheon 22012, Korea; [email protected] (J.S.B.); [email protected] (J.L.) 2 Department of Chemistry, Research Institute of Basic Sciences, Incheon National University, Incheon 22012, Korea * Correspondence: [email protected]; Tel.: +82-32-835-8094 These authors contribute equally to this article. y Received: 2 May 2019; Accepted: 18 June 2019; Published: 20 June 2019 Abstract: Recently, droplet-based microfluidic systems have been widely used in various biochemical and molecular biological assays. Since this platform technique allows manipulation of large amounts of data and also provides absolute accuracy in comparison to conventional bioanalytical approaches, over the last decade a range of basic biochemical and molecular biological operations have been transferred to drop-based microfluidic formats. In this review, we introduce recent advances and examples of droplet-based microfluidic techniques that have been applied in biochemistry and molecular biology research including genomics, proteomics and cellomics. Their advantages and weaknesses in various applications are also comprehensively discussed here. The purpose of this review is to provide a new point of view and current status in droplet-based microfluidics to biochemists and molecular biologists. We hope that this review will accelerate communications between researchers who are working in droplet-based microfluidics, biochemistry and molecular biology. Keywords: droplet-based microfluidic; biochemistry; molecular biology; digital PCR; biochip; biosensor; digital quantification; microfluidic; single cell analysis 1. -
The 'Omics Revolution: How an Obsession with Compiling Lists Is
The ‘omics revolution: how an obsession with compiling lists is threatening the ancient art of experimental design. Claudio D Stern Department of Cell & Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK. [email protected] The last quarter of a century ushered in a transformation in Biology following the huge successes in sequencing entire genomes of organisms (including humans) with increasing precision and speed and rapidly decreasing cost: genomics. Later other “omics” approaches followed: proteomics, metabolomics, epigenomics, and many more. Huge quantities of data were generated and new computational methods to find hidden correlations emerged. Almost imperceptibly, however, it seems that this has led to a crisis in our ability to design elegant experiments to establish causality. Imagine an extra-terrestrial excursion to planet Earth to discover about life on this planet. A spaceship with alien scientists lands in a park with many statues on display, representing important people from British history. The aliens unload their sophisticated analytical equipment and start to investigate. The first one uses a mass spectrometer – s/he approaches each statue and analyse it: one statue contains 5% copper, 10% iron, 17% aluminium, 63% lead. Another statue seems to consist mainly of carbon and a little water. A third object is made of granite. And so on. Another scientist follows: their analysis with a powerful super-resolution electron microscope reveals different sets of atomic structures. A third scientist uses a balance and lists the mass of each statue. A fourth measures the dimensions – some statues are very small, others quite large, and they have different overall shapes: some are tall, some are rather rounded. -
Technology Development for Single-Molecule Protein Sequencing
National Advisory Council for Human Genome Research May 18-19, 2020 Concept Clearance for RFA Technology Development for Single-Molecule Protein Sequencing Purpose: The purpose of this initiative is to accelerate innovation, development and early dissemination of single-molecule protein sequencing (SMPS) technologies. The ultimate goal is to achieve technological advances to the level where protein sequencing data can be generated at sufficient scale, speed, cost and accuracy to use routinely in studies of genome biology and function, and in biomedical research in general. This would enable analyses, such as deeper understanding of molecular phenotypes, identification of low abundance and ‘missing’ proteins, and true single-cell proteomics. This concept represents an ambitious and high-risk technology development challenge, and if successful, would provide a focused opportunity to transform the use of proteomics, in much the same way as modern next- generation nucleic acid sequencing (NGS) transformed genomics due to its high-throughput, low cost, and generalizability. Background: Through the Human Genome Project, and other efforts, NHGRI transformed biology by making genomics mainstream, with genomics now being a fundamental part of many studies of the biology of disease. Proteomics has not had the same widespread adoption, partially because of a lack of proteomics technologies that approach the scale of NGS, including improvements to the sensitivity and dynamic range of protein detection. The human proteome is extremely complex. A typical human cell expresses >10,000 unique protein gene products; and can contain ~100 times as many modified proteins, or proteoforms, for each gene product. In addition, the dynamic range of the proteome approaches seven orders of magnitude (from one copy per cell to ten million copies per cell) in tissues and cell lines, and up to ten orders of magnitude in blood plasma. -
From Proteomics to Systems Biology Outline and Learning Objectives
From Proteomics to Systems Biology Outline and learning objectives “Omics” science provides global analysis tools to study entire systems • How to obtain omics - data • What can we learn? Limitations? • Integration of omics - data • In-class practice: Omics-data visualization Integration of “omics”- information Omics - data provide systems-level information Omics - data provide systems-level information Whole-genome sequencing Microarrays 2D-electrophoresis, mass spectrometry Joyce & Palsson, 2006 Joyce & Palsson, 2006 Transcriptomics (indirectly) tells about Proteomics aims to detect and quantify RNA-transcript abundances a system’s entire protein content ! primary genomic readout Strengths: - very good genome-wide coverage - variety of commercial products Drawback: No protein-level info!! -> RNA degradation Strengths: -> Post-translational modifications -> info about post-translational modifications => validation by e.g. RT-PCR -> high throughput possible due to increasing quality and cycle speed of mass spec instrumentation Limitations: - coverage dependent on sample, preparation & separation method - bias towards most highly abundant proteins Omics - data provide systems-level information Metabolomics and Lipidomics Detector Metabolites GC-MS extracted NMR from cell lysate Glycan arrays, Lipids Glyco-gene chips, mass spec / NMR of carbohydrates ESI-MS/MS Joyce & Palsson, 2006 Metabolomics and Lipidomics Metabolomics and Lipidomics Metabolomics: Metabolomics: Large-scale measurement of cellular metabolites Large-scale measurement of -
Interfacing to Biological Systems Using Microfluidics
University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2018 Interfacing to Biological Systems Using Microfluidics Peter Golden Shankles University of Tennessee, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Recommended Citation Shankles, Peter Golden, "Interfacing to Biological Systems Using Microfluidics. " PhD diss., University of Tennessee, 2018. https://trace.tennessee.edu/utk_graddiss/5315 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Peter Golden Shankles entitled "Interfacing to Biological Systems Using Microfluidics." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Energy Science and Engineering. Scott T. Retterer, Major Professor We have read this dissertation and recommend its acceptance: Steven M. Abel, Mitchel J. Doctycz, Jennifer L. Morrell-Falvey Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Interfacing to Biological Systems Using Microfluidics A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Peter Golden Shankles December 2018 Copyright © 2018 by Peter Golden Shankles All rights reserved. -
Impacts of Genomics and Other 'Omics' for the Crop, Forestry, Livestock
Impacts of genomics and other ‘omics’ for the crop, forestry, livestock, fishery and agro-industry sectors in developing countries 1. Introduction Advances in genomics, the study of all the genetic material (i.e. the genome) of an organism, have been remarkable in recent years. Publication of the first draft of the human genome in 2001 was a milestone, quickly followed by that of the first crop (rice) in 2002 and the first farm animal (chicken) in 2004. Huge technological advancements have meant that sequencing has become dramatically quicker and cheaper over time, so the genomes of many of the important crops, livestock, forest trees, aquatic animals and agricultural pests are now already sequenced or soon will be. The FAO Biotechnology Forum (http://www.fao.org/biotech/biotech-forum/) is hosting this e-mail conference to look at the impacts that genomics, and the other related 'omics', have had so far on food and agriculture in developing countries as well as their potential impacts in the near future. Before looking at genomics in more detail, a quick overview of some basic genetic concepts can be provided [for more technical details, see FAO (2011a) or the FAO biotechnology glossary (FAO, 2001)]. All living things are made up of cells that contain genetic material called DNA, a molecule made up of a long chain of nitrogen-containing bases (of four kinds: A, C, G and T). DNA is organized as a double helix, where two DNA chains are held together through bonding of the bases, where A bonds with T and C bonds with G. -
Computational Proteomics: High-Throughput Analysis for Systems Biology
Pacific Symposium on Biocomputing 12:403-408(2007) COMPUTATIONAL PROTEOMICS: HIGH-THROUGHPUT ANALYSIS FOR SYSTEMS BIOLOGY WILLIAM CANNON Computational Biology & Bioinformatics, Pacific Northwest National Laboratory Richland, WA 99352 USA BOBBIE-JO WEBB-ROBERTSON Computational Biology & Bioinformatics, Pacific Northwest National Laboratory Richland, WA 99352, USA High-throughput proteomics is a rapidly developing field that offers the global profiling of proteins from a biological system. These high-throughput technological advances are fueling a revolution in biology, enabling analyses at the scale of entire systems (e.g., whole cells, tumors, or environmental communities). However, simply identifying the proteins in a cell is insufficient for understanding the underlying complexity and operating mechanisms of the overall system. Systems level investigations generating large-scale global data are relying more and more on computational analyses, especially in the field of proteomics. 1. Introduction Proteomics is a rapidly advancing field offering a new perspective to biological systems. As proteins are the action molecules of life, discovering their function, expression levels and interactions are essential to understanding biology from a systems level. The experimental approaches to performing these tasks in a high- throughput (HTP) manner vary from evaluating small fragments of peptides using tandem mass spectrometry (MS/MS), to two-hybrid and affinity-based pull-down assays using intact proteins to identify interactions. No matter the approach, proteomics is revolutionizing the way we study biological systems, and will ultimately lead to advancements in identification and treatment of disease as well as provide a more fundamental understanding of biological systems. The challenges however are amazingly diverse, ranging from understanding statistical models of error in the experimental processes through categorization of tissue types. -
New Technologies and Their Impact on 'Omics' Research
Available online at www.sciencedirect.com New technologies and their impact on ‘omics’ research Editorial overview Matthew Bogyo and Pauline M Rudd Current Opinion in Chemical Biology 2013, 17:1–3 For a complete overview see the Issue 1367-5931/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.cbpa.2013.01.005 Matthew Bogyo The use of the suffix ‘ome’ is defined in the Oxford English dictionary as ‘being used in cellular and molecular biology to form nouns with the sense Department of Pathology, Stanford University ‘‘all constituents considered collectively’’’. Interestingly, the use of the School of Medicine, Stanford, CA, USA e-mail: [email protected] ‘ome’ suffix in biology dates back to the early 1900s when it was first used to describe a ‘biome’ and genome (although originally in German as genom). Matthew Bogyo received his BSc degree in Over the past few decades, the use of the omics suffix has rapidly increased, Chemistry from Bates College in 1993 and a due in a large part, to the rapid growth in technologies that allow global PhD in Biochemistry from the Massachusetts analysis of samples on a systems level. The familiarity of the ‘omics’ term Institute of Technology in 1997. He became a also was rapidly advanced by the completion of the human genome in the Faculty Fellow at the University of California, early 2000s. Since that time, we have seen the term ‘omics’ move beyond use San Francisco in 1998. In 2001, he moved to for genomics and proteomics and gain acceptance for a diverse array of Celera Genomics to head the Department of Chemical Proteomics until 2003 when he systems biology applications. -
A Flexible Microfluidic System for Single-Cell Transcriptome Profiling
www.nature.com/scientificreports OPEN A fexible microfuidic system for single‑cell transcriptome profling elucidates phased transcriptional regulators of cell cycle Karen Davey1,7, Daniel Wong2,7, Filip Konopacki2, Eugene Kwa1, Tony Ly3, Heike Fiegler2 & Christopher R. Sibley 1,4,5,6* Single cell transcriptome profling has emerged as a breakthrough technology for the high‑resolution understanding of complex cellular systems. Here we report a fexible, cost‑efective and user‑ friendly droplet‑based microfuidics system, called the Nadia Instrument, that can allow 3′ mRNA capture of ~ 50,000 single cells or individual nuclei in a single run. The precise pressure‑based system demonstrates highly reproducible droplet size, low doublet rates and high mRNA capture efciencies that compare favorably in the feld. Moreover, when combined with the Nadia Innovate, the system can be transformed into an adaptable setup that enables use of diferent bufers and barcoded bead confgurations to facilitate diverse applications. Finally, by 3′ mRNA profling asynchronous human and mouse cells at diferent phases of the cell cycle, we demonstrate the system’s ability to readily distinguish distinct cell populations and infer underlying transcriptional regulatory networks. Notably this provided supportive evidence for multiple transcription factors that had little or no known link to the cell cycle (e.g. DRAP1, ZKSCAN1 and CEBPZ). In summary, the Nadia platform represents a promising and fexible technology for future transcriptomic studies, and other related applications, at cell resolution. Single cell transcriptome profling has recently emerged as a breakthrough technology for understanding how cellular heterogeneity contributes to complex biological systems. Indeed, cultured cells, microorganisms, biopsies, blood and other tissues can be rapidly profled for quantifcation of gene expression at cell resolution. -
Molecular Biologist's Guide to Proteomics
Molecular Biologist's Guide to Proteomics Paul R. Graves and Timothy A. J. Haystead Microbiol. Mol. Biol. Rev. 2002, 66(1):39. DOI: 10.1128/MMBR.66.1.39-63.2002. Downloaded from Updated information and services can be found at: http://mmbr.asm.org/content/66/1/39 These include: REFERENCES This article cites 172 articles, 34 of which can be accessed free http://mmbr.asm.org/ at: http://mmbr.asm.org/content/66/1/39#ref-list-1 CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» on November 20, 2014 by UNIV OF KENTUCKY Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2002, p. 39–63 Vol. 66, No. 1 1092-2172/02/$04.00ϩ0 DOI: 10.1128/MMBR.66.1.39–63.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. Molecular Biologist’s Guide to Proteomics Paul R. Graves1 and Timothy A. J. Haystead1,2* Department of Pharmacology and Cancer Biology, Duke University,1 and Serenex Inc.,2 Durham, North Carolina 27710 INTRODUCTION .........................................................................................................................................................40 Definitions..................................................................................................................................................................40 Downloaded from Proteomics Origins ...................................................................................................................................................40 -
Clinical Proteomics
Clinical Proteomics Michael A. Gillette Broad Institute of MIT and Harvard Massachusetts General Hospital “Clinical proteomics” encompasses a spectrum of activity from pre-clinical discovery to applied diagnostics • Proteomics applied to clinically relevant materials – “Quantitative and qualitative profiling of proteins and peptides that are present in clinical specimens like human tissues and body fluids” • Proteomics addressing a clinical question or need – Discovery, analytical and preclinical validation of novel diagnostic or therapy related markers • MS-based and/or proteomics-derived test in the clinical laboratory and informing clinical decision making – Clinical implementation of tests developed above – Emphasis on fluid proteomics – Includes the selection, validation and assessment of standard operating procedures (SOPs) in order that adequate and robust methods are integrated into the workflow of clinical laboratories – Dominated by the language of clinical chemists: Linearity, precision, bias, repeatability, reproducibility, stability, etc. Ref: Apweiler et al. Clin Chem Lab Med 2009 MS workflow allows precise relative quantification of global proteome and phosphoproteome across large numbers of samples Tissue, cell lines, biological fluids 11,000 – 12,000 distinct proteins/sample 25,000 - 30,000 phosphosites/sample Longitudinal QC analyses of PDX breast cancer sample demonstrate stability and reproducibility of complex analytic workflow Deep proteomic and phosphoproteomic annotation for 105 genomically characterized TCGA breast -
Important Considerations for Sample Collection in Metabolomics Studies with a Special Focus on Applications to Liver Functions
H OH metabolites OH Review Important Considerations for Sample Collection in Metabolomics Studies with a Special Focus on Applications to Liver Functions Lorraine Smith 1, Joran Villaret-Cazadamont 1, Sandrine P. Claus 2 ,Cécile Canlet 1, Hervé Guillou 1 , Nicolas J. Cabaton 1 and Sandrine Ellero-Simatos 1,* 1 Toxalim (Research Center in Food Toxicology), Université de Toulouse, INRAE, ENVT, INP-Purpan, UPS, 31300 Toulouse, France; [email protected] (L.S.); [email protected] (J.V.-C.); [email protected] (C.C.); [email protected] (H.G.); [email protected] (N.J.C.) 2 LNC Therapeutics, 17 place de la Bourse, 33076 Bordeaux, France; [email protected] * Correspondence: [email protected] Received: 11 February 2020; Accepted: 7 March 2020; Published: 12 March 2020 Abstract: Metabolomics has found numerous applications in the study of liver metabolism in health and disease. Metabolomics studies can be conducted in a variety of biological matrices ranging from easily accessible biofluids such as urine, blood or feces, to organs, tissues or even cells. Sample collection and storage are critical steps for which standard operating procedures must be followed. Inappropriate sample collection or storage can indeed result in high variability, interferences with instrumentation or degradation of metabolites. In this review, we will first highlight important general factors that should be considered when planning sample collection in the study design of metabolomic studies, such as nutritional status and circadian rhythm. Then, we will discuss in more detail the specific procedures that have been described for optimal pre-analytical handling of the most commonly used matrices (urine, blood, feces, tissues and cells).