DOCSLIB.ORG

UNIVERSITY of ALBERTA High Sensitivity Protein Sequencing

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
UNIVERSITY of ALBERTA High Sensitivity Protein Sequencing UNIVERSITY OF ALBERTA High Sensitivity Protein Sequencing using Fluorescein Isothiocyanate for Denvatization on a Miniaturized Sequencer using Capillary EIectrophoresis with Laser hduced Fluorescence Detection for Prduct Identification. A thesis submitted to the Faculty of Graduate Studies and Research in partial Mfiment of the requirernents for the degree of Doctor of Philosophy. DEPARTMENT OF CHEMISTRY EDMONTON, ALBERTA, CANADA SPRING 2000 National Library Bibliothèque nationale 141 ,cm,, du Canada Acquisitions and Acquisitions et Bibtiographic Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada The author has granted a non- L'auteur a accordé une Licence non exclusive licence allowing the exclusive permettant à la National Lïbrary of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or selI reproduire, prêter, distribuer ou copies of this thesis in rnicroform, vendre des copies de cette thèse sous paper or electronic formats. la fonne de microfiche/fïlm, de reproduction sur papier ou sur format électronique. The author retauis ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. For my Parents Primary sequence malysis is an important step in determining the strùctr;re- function relationship for a @en protein or peptide. By far the most popular sequencing method in use today is the Edman degradation. A number of automated Edman sequencers are commerciaily available. They dl incorporate high perto rmance Liquid chro matography (HPLC) with ultraviolet (UV) absorbance detection for identification of the phenykhiohydantoin (PTK) amino acid sequencing products. This detection system is suitable for the analysis of proteins at the low picornole level. However, many biologically active proteins can only be isolated at the mid to low femtomole level. This thesis describes the development of a sequencer that has the potential to successfully analyse these rare proteins. The new sequencer incorporates three major modifications from current instruments. First. sequencing products are identified by capillary eIectrophoresis with laser induced fluorescence (CE-LIF) detection. This approzch allows for unambiguous identification of the fluorescein thiohydantoin (FTH) derivatives of the eighteen coded amino acids studied thus far- Furthemore, mass limits of detection are in the low zeptomole (lzmol= Lx~o-" mols) range for all components in the separation. This sensitivity is seven to eight orders of magnitude better than that of HPLC with W absorbance detection. The second modification involves a change tiom phenylisothiocyanate (PITC) to fluorescein isothiocyanaie (FITC) as the prirnary derivatizing agent in the Edman degradation. It is this change that resuIts in formation of the FTH-amino acid sequencing products that can be detected with such exquisite sensitivity by CE-LIF. Finaliy, the sequencer has been miniaturized io make it more compatible with the volume requirements of CE. Reagents are deiivered by syringe pumps to a reaction chamber that is a traction of the sue of those hund on commercial sequencers- This thesis presents data describing the CE-LIF separation of 18 FTH-amino acids. It then descnbes the syringe pump sequencer and the modified Edrnan degradation chemistry. It fmishes by presenting sequencing data for five cycles of P-lactoglobulin at the 25 pmol. 10 pmol and 1 pmol levels. The data shows that this proof of p~ciple instrument already has a sensitivity comparable to that of state of the art Edman sequencers. Preface 16 GENERATIONS OF SCIENCE PERSON SCHOOL DATE Ian D. Ireland Alberta 1999 Norman J. Dovichi Utah 1980 Joel M. Harris Purdue 1976 Fred E. Lytle M.I.T. 1968 David M. Hercules MIT. 1957 Lockhart B. Rogers Princeton 1942 Earle C. Caley Ohio State 1928 Charles W. Fauik Leipzig 1890 Wilhelm Ostwald Dorpat 1878 Car1 Schmidt Giessen 1844 Justus von Liebig Er1angen 1822 Joseph L. Gay-Lussac Paris 1800 Claude L, Berthollet Pans 1778 Jean B.M. Bucquet Paris 1770 Antoine L. Lavoisier Pans 1763 GuiIIaume F. Rouelle Paris Apothecary School 1725 Norther Lights Laser Lab Ian Ireland - 1999 Rong Jiang - 1998 Devanand Pinto - 1998 Sue Bay - 1998 Karl Voss - 1998 Darcy Hager - 1997 H. John Crabtree - 1996 Yanni Zhang - 1996 Daniel Figeys - 1995 Heather Starke - 1994 JianYing Zhao - 1994 JianZhong Zhang - 1994 Karen Waldron - 1993 DaYong Chen - 1993 Robert McLaren - 199 1 YungFong Cheng - 1988 Danyl Bornhop - 1987 Dean Burgi - 1987 Tomas Nolan - 1986 Wayne Weimer - 1985 Fahima Zarrïn - 1985 Obtaining a Ph.D. in any field involves a huge cornmitment in both the and energy. And, as with any such endeavor, its successful completion requires support, both practical and emotional, from a large nuniber of people. In the next few paragraphs, 1 would like to express rny appreciation to those people who helped me to reach such a meaningful personal rnilestone. First, 1would like to express my heartfelt thanks to Nom Dovichi, my supervisor. Norm, when 1 made the decision to join the Northern Lights Laser Lab, 1 was attracted not ody by the wonderful science being done in the group but also by your genuine interest in and concern for the people that you supervise. 1 know that if I had to do it over again (heaven forbid), 1 would unhesitatingly make the same choice that 1 made almost seven (!) years ago. 1 would also like to thank al1 of the other mernbers of the NLLL with whom 1 had the pleasure of working. There always seemed to be someone in the group who had the expertise to help with any problem that arose. In particular, 1 wodd like to thank Xing- Fang Li and Darren Lewis for al1 of the work they did on the design and construction of the sequencer and Min Chen and Annika Renborg for their help with the FTH-amino acid separation. There is another group in the Chernistry Department who played a large part in the success of this project. These people work in the machine, gIassblowing and electronics shops. They were responsible for making the various sequencer components. Their input went beyond simply constructing the components. They often suggested design modifications that led to significant irnprovements to the finished instrument. Some of the people who helped me the most while 1 was at the University of Alberta were not directly involved in the development of the protein sequencer. Instead, they provided the encouragement and emotionai support outside of the lab that kept my spirits up during the bad times and made the good times that much better. Lanssa Powell carne to Edmonton with me. For more than five years we made a home together and shared our hopes and drearns. Larissa, things didn't turn out the way we planned, but 1 know that you will be successful, both at work and at home. 1 am grateful to Lorraine and Roland Zimmermann (and the boys) for their unwavering support. They gave me a place to go on those days when 1 needed to forget my worries and just escape for a while. Lorraine, it was a pleasure working with you and I'm glad that you are back doing something you enjoy. 1am also deeply indebted to the McDemott farnily - Mark, Christie, Jordan and Quentin. You opened up your home to me (not to mention your fridge and your liquor cabinet) and made me feel Iike I was part of the family. For that 1 wiil dways be grateful. 1 carmot adequately express how much your understanding and encouragement have meant to me. Finally, 1 corne to my parents and my sister, Vikki - my fdy. You have always been there for me and you know me the best. It would take pages to descnbe all that you have done for me. Instead, 1 will content myself with two heartfelt thoughts: thank you and I love you. Table of Contents 1 Introduction ........................................................................................................... 1 1. 1 Introduction............................................................................................. 1 1-2 Capillary Electro p horesis ................................................................... 2 1.3 Laser Induced Fluorescence Detection.................................................... -21 1.4 Protein Sequencing .................................................................................. 34 1-5 References............................................................................................ 56 Appendk A: L-oc-amino acid structures ........................................................... 62 2 Identification of Fluorescein Thiohydantoin Amino Acids by Capillary Electrophoresis with Laser Induced Fluorescence Detection as a Potentiai Method for Improving Sensitivity in Protein Sequencing ................................................. ... 63 Introduction............................................................................................. 63 Experirnental ..................................................................................... 65 1.2.1 Fluorescein Thiohydantoin Amino Acid Synthesis ................... -65 1.2.2 Capillary Electro phoresis wit h Laser Induced Fluorescence Detection .................................................................................. 66 Results and Discussion ..........................................................................
Load more

Recommended publications
  • Protein Sequencing Production of Ions for Mass Spectrometry
    Lecture 1 Introduction- Protein Sequencing Production of Ions for Mass Spectrometry Nancy Allbritton, M.D., Ph.D. Department of Physiology & Biophysics 824-9137 (office) [email protected] Office- Rm D349 Medical Science D Bldg. Introduction to Proteins Amino Acid- structural unit of a protein α carbon Amino acids- linked by peptide (amide) bond Amino Acids Proteins- 20 amino acids (Recall DNA- 4 bases) R groups- Varying size, shape, charge, H-bonding capacity, & chemical reactivity Introduction to Proteins Polypeptide Chain (Protein) - Many amino acids linked by peptide bonds By convention: Residue 1 starts at amino terminus. Polypeptides- a. Main chain i.e. regularly repeating portion b. Side chains- variable portion Introduction to Proteins 25,000 human genes >2X106 proteins Natural Proteins - Typically 50-2000 amino acids i.e. 550-220,000 molecular weight Over 200 different types of post-translational modifications. Ex: proteolysis, phosphorylation, acetylation, glycosylation S S S S S S Ex: Insulin Protein Complexity Is Very Large Over 200 different types of post-translational modifications. The Problem of Protein Sequencing. Edman Degradation: Step-wise cleavage of an amino acid from the amino terminus of a peptide. Alanine Gly 1 2 Reacts with uncharged NH3 1 2 Gly 1 2 Cyclizes & Releases in Mild Acid H N-Gly 2 2 2 PTH-Alanine Edman Degradation 1. Must be short peptide (<50 a.a.) amino acid release- 98% efficiency proteins- must fragment (CNBr or trypsin) Trypsin 2. Frequently fails due to a blocked amino terminus 3. Intolerant of impurities 4. Tedious & time consuming (hours-days) 1 amino acid cycle- 2 hours Solution: Mass Spectrometry 1.
    [Show full text]
  • 9 3 Innovirolgy Examples and Applications
    9.3: Examples and applications 1) Now that you have an understanding of the mechanisms behind the phage display system, let’s have a look at how it’s used in research. i) Phage display has a broad spectrum of applications. This can go from improvement of enzymes, to proteome analysis or drug and target discovery but is certainly not restricted to these. 2) The first thing I want to focus on is the optimization of enzymes. A large variety of enzymes are being used today in different fields of industry and biotech. These enzymes are originally made by organisms for a specific function in a specific niche or environment, like inside the cell cytoplasm. Therefore, they often have undesirable properties for the function or environment we want to use them in. They might not work fast enough, be unstable in the conditions they are used in or do not work on the desired substrate. Phage display can aid in improving these enzymes by selecting for example for better thermostability, activity or enhanced substrate binding. i) Let’s start with stability. Take for example an enzyme that is normally present In a cell at cytoplasm at 37°C. However when we use it in an industrial process where we need 50°C, the enzyme becomes unstable and denatures. In phage display we can make a library of mutagenized enzyme and display them on, in this case, the gene 3 protein of the phage. In the binding step of the biopanning most phages will not be able to bind the enzyme substrate at 50°C as they are denatured.
    [Show full text]
  • Single-Molecule Peptide Fingerprinting
    Single-molecule peptide fingerprinting Jetty van Ginkela,b, Mike Filiusa,b, Malwina Szczepaniaka,b, Pawel Tulinskia,b, Anne S. Meyera,b,1, and Chirlmin Joo (주철민)a,b,1 aKavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands; and bDepartment of Bionanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands Edited by Alan R. Fersht, Gonville and Caius College, Cambridge, United Kingdom, and approved February 21, 2018 (received for review May 1, 2017) Proteomic analyses provide essential information on molecular To obtain ordered determination of fluorescently labeled amino pathways of cellular systems and the state of a living organism. acids, we needed a molecular probe that can scan a peptide in a Mass spectrometry is currently the first choice for proteomic processive manner. We adopted a naturally existing molecular analysis. However, the requirement for a large amount of sample machinery, the AAA+ protease ClpXP from Escherichia coli.The renders a small-scale proteomics study challenging. Here, we ClpXP protein complex is an enzymatic motor that unfolds and demonstrate a proof of concept of single-molecule FRET-based degrades protein substrates. ClpX monomers form a homohexa- + protein fingerprinting. We harnessed the AAA protease ClpXP to meric ring (ClpX6) that can exercise a large mechanical force to scan peptides. By using donor fluorophore-labeled ClpP, we se- unfold proteins using ATP hydrolysis (11, 12). Through iterative quentially read out FRET signals from acceptor-labeled amino acids rounds of force-generating power strokes, ClpX6 translocates of peptides. The repurposed ClpXP exhibits unidirectional process- substrates through the center of its ring in a processive manner ing with high processivity and has the potential to detect low- (13, 14), with extensive promiscuity toward unnatural substrate abundance proteins.
    [Show full text]
  • Intrinsic Indicators for Specimen Degradation
    Laboratory Investigation (2013) 93, 242–253 & 2013 USCAP, Inc All rights reserved 0023-6837/13 $32.00 Intrinsic indicators for specimen degradation Jie Li1, Catherine Kil1, Kelly Considine1, Bartosz Smarkucki1, Michael C Stankewich1, Brian Balgley2 and Alexander O Vortmeyer1 Variable degrees of molecular degradation occur in human surgical specimens before clinical examination and severely affect analytical results. We therefore initiated an investigation to identify protein markers for tissue degradation assessment. We exposed 4 cell lines and 64 surgical/autopsy specimens to defined periods of time at room temperature before procurement (experimental cold ischemic time (CIT)-dependent tissue degradation model). Using two-dimen- sional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, we performed comparative proteomic analyses on cells at different CIT exposures and identified proteins with CIT-dependent changes. The results were validated by testing clinical specimens with western blot analysis. We identified 26 proteins that underwent dynamic changes (characterized by continuous quantitative changes, isoelectric changes, and/or proteolytic cleavages) in our degradation model. These changes are strongly associated with the length of CIT. We demonstrate these proteins to represent universal tissue degradation indicators (TDIs) in clinical specimens. We also devised and implemented a unique degradation measure by calculating the quantitative ratio between TDIs’ intact forms and their respective degradation-
    [Show full text]
  • M.Sc. [Botany] 346 13
    cover page as mentioned below: below: mentioned Youas arepage instructedcover the to updateupdate to the coverinstructed pageare asYou mentioned below: Increase the font size of the Course Name. Name. 1. IncreaseCourse the theof fontsize sizefont ofthe the CourseIncrease 1. Name. use the following as a header in the Cover Page. Page. Cover 2. the usein the followingheader a as as a headerfollowing the inuse the 2. Cover Page. ALAGAPPAUNIVERSITY UNIVERSITYALAGAPPA [Accredited with ’A+’ Grade by NAAC (CGPA:3.64) in the Third Cycle Cycle Third the in (CGPA:3.64) [AccreditedNAAC by withGrade ’A+’’A+’ Gradewith by NAAC[Accredited (CGPA:3.64) in the Third Cycle and Graded as Category–I University by MHRD-UGC] MHRD-UGC] by University and Category–I Graded as as Graded Category–I and University by MHRD-UGC] M.Sc. [Botany] 003 630 – KARAIKUDIKARAIKUDI – 630 003 346 13 EDUCATION DIRECTORATEDISTANCE OF OF DISTANCEDIRECTORATE EDUCATION BIOLOGICAL TECHNIQUES IN BOTANY I - Semester BOTANY IN TECHNIQUES BIOLOGICAL M.Sc. [Botany] 346 13 cover page as mentioned below: below: mentioned Youas arepage instructedcover the to updateupdate to the coverinstructed pageare asYou mentioned below: Increase the font size of the Course Name. Name. 1. IncreaseCourse the theof fontsize sizefont ofthe the CourseIncrease 1. Name. use the following as a header in the Cover Page. Page. Cover 2. the usein the followingheader a as as a headerfollowing the inuse the 2. Cover Page. ALAGAPPAUNIVERSITY UNIVERSITYALAGAPPA [Accredited with ’A+’ Grade by NAAC (CGPA:3.64) in the Third Cycle Cycle Third the in (CGPA:3.64) [AccreditedNAAC by withGrade ’A+’’A+’ Gradewith by NAAC[Accredited (CGPA:3.64) in the Third Cycle and Graded as Category–I University by MHRD-UGC] MHRD-UGC] by University and Category–I Graded as as Graded Category–I and University by MHRD-UGC] M.Sc.
    [Show full text]
  • Electrophoresis
    B2144 Cover.qxd 9/1/98 8:10 AM Page 2 Ready Gel System RESOURCE GUIDE Bio-Rad Laboratories Life Science Australia, Bio-Rad Laboratories Pty Limited, Block Y Unit 1, Regents Park Industrial Estate, 391 Park Road, Regents Park, NSW 2143 • Group Phone 02-9914-2800 • Fax 02-9914-2889 Austria, Bio-Rad Laboratories Ges.m.b.H., Auhofstrasse 78D, A-1130 Wien • Phone (1) 877 89 01 • Fax (1) 876 56 29 Belgium, Bio-Rad Laboratories S.A.-N.V., Begoniastraat 5, B-9810 Nazareth EKE • Phone 09-385 55 11 • Fax 09-385 65 54 2000 Alfred Nobel Drive Canada, Bio-Rad Laboratories (Canada) Ltd., 5671 McAdam Road, Mississauga, Ontario L4Z 1N9 • Phone (905) 712-2771 • Fax (905) 712-2990 Hercules, California 94547 China, Bio-Rad China (Beijing), 14 Zhi Chun Road, Hai Dian District, Beijing 100 008 • Phone 86-10-62051850 • Fax 86-10-62051876 Telephone (510) 741-1000 Denmark, Bio-Rad Laboratories, Symbion Science Park, Fruebjergvej 3, DK-2100 København Ø • Phone 39 17 9947 • Fax 39 27 1698 Fax: (510) 741-5800 Finland, Bio-Rad Laboratories, Pihatörmä 1A 02240, Espoo, • Phone 90 804 2200 • Fax 90 804 1100 www.bio-rad.com France, Bio-Rad S.A., 94/96 rue Victor Hugo, B.P. 220, 94 203 Ivry Sur Seine Cedex • Phone (1) 43 90 46 90 • Fax (1) 46 71 24 67 Germany, Bio-Rad Laboratories GmbH, Heidemannstraße 164, D-80939 München • Phone 089 31884-0 • Fax 089 31884-100 Hong Kong, Bio-Rad Pacific Ltd., Unit 1111, 11/F., New Kowloon Plaza, 38 Tai Kok Tsui Road, Tai Kok Tsui, Kowloon, Hong Kong • Phone 852-2789-3300 • Fax 852-2789-1257 India, Bio-Rad Laboratories, India (Pvt.) Ltd., C-248 Defence Colony, New Delhi 110 024 • Phone 91-11-461-0103 • Fax 91-11-461-0765 Israel, Bio-Rad Laboratories Ltd., 12 Homa Street, P.O.
    [Show full text]
  • De Novo Peptide Sequencing Tutorial
    http://ionsource.com/tutorial/DeNovo/introduction.htm - De Novo Peptide Sequencing Tutorial 1 http://ionsource.com/tutorial/DeNovo/introduction.htm Table of Contents Introduction - Peptide Fragmentation Nomenclature - b and y Ions - The Rules (16 simple rules) - The Protocol - Example MS/MS Spectrum - • low mass • mid mass • high mass - First De Novo Exercise (Ion Trap Data) - • Answer Page - Second De Novo Exercise (q-TOF Data) - • Answer page • Tutorial References and Additional Suggested Reading - • De Novo Tables - AA Residue Mass, Mass Conflict, Di-peptide Mass Residue Combinations 2 http://ionsource.com/tutorial/DeNovo/introduction.htm 3 http://ionsource.com/tutorial/DeNovo/introduction.htm Introduction De novo is Latin for, "over again", or "anew". A popular definition for "de novo peptide sequen- cing" is, peptide sequencing performed without prior knowledge of the amino acid sequence. Usually this rule is imposed by Edman degradation practitioners who perform de novo sequencing day in and day out, and perhaps feel a little bit threatened by that half million dollar mass spectrometer sitting down the hall, that can supposedly sequence peptides in a matter of seconds, and not days. Actually, any research project should be started with as much information as pos- sible, there should never be a need to restrict your starting knowledge, unless of course you are performing a clinical trial or some other highly controlled experiment. Mass spectrometers do have the advantage when it comes to generating sequence data for peptides in low femtomole quantities. However, Edman degradation will always enjoy the advantage when pmol quantities of a peptide are available. At higher pmol quantities (2-10 pmol), Edman will often provide the exact amino acid sequence without ambiguity for a limited run of amino acids, 6-30 amino acids, usually taking 30-50 min per cycle of the sequencer.
    [Show full text]
  • Mass Spectrometry-Based De Novo Sequencing of the Anti-FLAG-M2 Antibody Using 2 Multiple Proteases and a Dual Fragmentation Scheme
    bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425675; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Mass spectrometry-based de novo sequencing of the anti-FLAG-M2 antibody using 2 multiple proteases and a dual fragmentation scheme 3 4 Authors: 5 Weiwei Peng1#, Matti F. Pronker1#, Joost Snijder1* 6 7 #equal contribution 8 *corresponding author: [email protected] 9 10 Affiliation: 11 1 BioMolecular Mass SpectroMetry and ProteoMics, Bijvoet Center for BioMolecular Research 12 and Utrecht Institute of PharMaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH 13 Utrecht, The Netherlands 14 15 Keywords: 16 mass spectrometry, antibody, de novo sequencing, EThcD, stepped HCD, Herceptin, FLAG tag, 17 anti-FLAG-M2. 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425675; this version posted January 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 18 Abstract: 19 Antibody sequence inforMation is crucial to understanding the structural basis for antigen binding 20 and enables the use of antibodies as therapeutics and research tools. Here we deMonstrate a 21 method for direct de novo sequencing of Monoclonal IgG from the purified antibody products.
    [Show full text]
  • Analysis of Transcript and Protein Overlap in a Human Osteosarcoma Cell Line
    Klevebring et al. BMC Genomics 2010, 11:684 http://www.biomedcentral.com/1471-2164/11/684 RESEARCH ARTICLE Open Access Analysis of transcript and protein overlap in a human osteosarcoma cell line Daniel Klevebring1,3, Linn Fagerberg2, Emma Lundberg2, Olof Emanuelsson1, Mathias Uhlén2, Joakim Lundeberg1* Abstract Background: An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high- throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq) to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC) and immunofluorescence microscopy (IF). Results: A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2%) were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis.
    [Show full text]
  • Protein Detection & Identification Methods
    Protein Detection & Identification Methods October 24, 2007 MSB B554 Hong Li [email protected] 973-972-8396 Lecture notes: http://njms.umdnj.edu/proweb/lectures/note2007fall01.pdf Objectives 1. Protein analysis to determine: • Purity, quantity and identity • Expression and localization • Post-translational modification • Induction and turnover 2. Principles behind the analytical techniques • Based on unique physical/chemical properties; size, charge, etc. • Assays are based on reactions producing light, color and radio activities for detection 3. Techniques • Electrophoresis • Immunoblotting • Autoradiography • Mass spectrometry • Proteomics 1 Purity, quantity and identity Post-translational modification Induction and turnover Expression localization Basic Principles for Analysis (How to differentiate one protein from another?) Structure (Drs. Wang & Wah) Amino acid composition Post-translational modification (Dr. Wagner) Size Polarity/Charge/Hydrophobicity Shape Affinity (binding to other proteins/molecules) Function: catalytic activities 2 Shapes and sizes # of amino acids, composition & sequences Charges and polarity 3 Charges and polarity from post-translational modifications Reactivities of amino acids Physical/chemical reactions to facilitate colorimetric detection Example: Protein concentration assays: Bradford, BCA, Lowry & Biuret, etc. http://www-class.unl.edu/biochem/protein_assay/ 4 Bradford assay (Bio-Rad) Based on a dye binding to basic and aromatic amino acids Coommassie Brilliant Blue (CBB) G250 Protein binding
    [Show full text]
  • Green/Red Cyanobacteriochromes Regulate Complementary Chromatic Acclimation Via a Protochromic Photocycle
    Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle Yuu Hirosea, Nathan C. Rockwellb, Kaori Nishiyamac, Rei Narikawad, Yutaka Ukajic, Katsuhiko Inomatac, J. Clark Lagariasb,1, and Masahiko Ikeuchid,1 aElectronics-Inspired Interdisciplinary Research Institute, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan; bDepartment of Molecular and Cellular Biology, University of California, Davis, CA 95616; cDivision of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan; and dDepartment of Life Sciences (Biology), The University of Tokyo, Meguro, Tokyo 153-8902, Japan Contributed by J. Clark Lagarias, February 13, 2013 (sent for review January 24, 2013) Cyanobacteriochromes (CBCRs) are cyanobacterial members of the spectral diversity, with peak absorptions ranging from 330 to 680 phytochrome superfamily of photosensors. Like phytochromes, nm and hence spanning the entire visible spectrum and near UV CBCRs convert between two photostates by photoisomerization (13, 15–24). CBCR subfamilies that sense light in the near-UV to of a covalently bound linear tetrapyrrole (bilin) chromophore. blue region (330–470 nm) have been studied extensively. Such Although phytochromes are red/far-red sensors, CBCRs exhibit CBCRs combine bilin photoisomerization with subsequent for- diverse photocycles spanning the visible spectrum and the near- mation or elimination of a second thioether linkage to the bilin UV (330–680 nm). Two CBCR subfamilies detect near-UV to blue light C10 atom, shortening the conjugated π system to induce a re- (330–450 nm) via a “two-Cys photocycle” that couples bilin 15Z/15E markable spectral shift (19, 21, 23, 25–29). In such “two-Cys photoisomerization with formation or elimination of a second bilin– photocycles,” the reactive thiol group is supplied by a second cysteine adduct.
    [Show full text]
  • The Development of the Prediction of Protein Structure
    6 The Development of the Prediction of Protein Structure Gerald D. Fasman I. Introduction .................................................................... 194 II. Protein Topology. .. 196 III. Techniques of Protein Prediction ................................................... 198 A. Sequence Alignment .......................................................... 199 B. Hydrophobicity .............................................................. 200 C. Minimum Energy Calculations ................................................. 202 IV. Approaches to Protein Conformation ................................................ 203 A. Solvent Accessibility ......................................................... 203 B. Packing of Residues .......................................................... 204 C. Distance Geometry ........................................................... 205 D. Amino Acid Physicochemical Properties ......................................... 205 V. Prediction of the Secondary Structure of Proteins: a Helix, ~ Strands, and ~ Turn .......... 208 A. ~ Turns .................................................................... 209 B. Evaluation of Predictive Methodologies .......................................... 218 C. Other Predictive Algorithms ................................................... 222 D. Chou-Fasman Algorithm ...................................................... 224 E. Class Prediction ............................................................. 233 VI. Prediction of Tertiary Structure ...................................................
    [Show full text]