Carbohydrate Structure Data Base for Capillary Electrophoresis Profile Allocation  Page description

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Details of project

  
Identifier
81839
Type K
Principal investigator Guttman, András
Title in Hungarian Szénhidrát-szerkezeti adatbázis létrehozása kapilláris elektroforézisen alapuló eredmények alapján
Title in English Carbohydrate Structure Data Base for Capillary Electrophoresis Profile Allocation
Keywords in Hungarian glikán, kapilláris elektroforézis, tömegspektrometria, adatbázis, szerkezetmeghatározás
Keywords in English glycan, capillary electrophoresis, mass spectrometry, database, structural analysis
Discipline
Analytical Chemistry (Council of Physical Sciences)60 %
Ortelius classification: Analytical chemistry
Bioinformatics (Council of Medical and Biological Sciences)40 %
Panel Chemistry 1
Department or equivalent Research Centre for Molecular Medicine (University of Debrecen)
Participants Kovács, András
Mittermayr, Stefan
Starting date 2010-09-01
Closing date 2013-08-31
Funding (in million HUF) 28.400
FTE (full time equivalent) 6.73
state closed project
Summary in Hungarian
A glikánok szerkezet-meghatározása nem könnyű feladat, mivel struktúráik és szintézisútjaik nagyon változatosak, ugyanakkor az oligoszacharidok fizikai és kémiai tulajdonságai eléggé hasonlóak. A lézer indukált detektálással kapcsolt kapilláris elektroforézis (CE-LIF) kiválóan alkalmas analitikai módszer a fluoreszcensen jelzett glikánok vizsgálatára, beleértve a pozicionális és kötési izomereket is. Annak ellenére, hogy a CE-LIF-fel végzett elválasztások hatásfoka és reszolúciója jobb, és a szeparációs idők rövidebbek, mint a HPLC esetében, jelenleg nincsenek a migrációs időket és glükózegység-értékeket (GU) tartalmazó szerkezeti adatbázisok. Egy átfogó, kapilláris elektroforézis-eredményeket és GU-értékeket felölelő adatbázis létrehozása nagyban könnyítené a glikánok mélyebb szerkezeti azonosítását. Ennek érdekében tervezzük egy, nagyszámú N-glikán tulajdonságait tartalmazó adatbázis létrehozását, három különböző oligoszacharid-létra segítségével: (Glcα1,4)n, (Glcβ1,4)n és (GlcNAc)n. Azt várjuk, hogy a detektált glikánok többségét szerkezeti tulajdonságaik - GU-értékeik - alapján osztályokba tudjuk majd sorolni. Mivel különböző struktúrájú glikánok migrációs értékei azonosak lehetnek, exoglikozidázos emésztés és tömegspektrometriai elemzés is szükséges lesz. A kapott glikán-adatbázis egy felhasználóbarát webfelületen nyilvánosan elérhető lesz, így a kapilláris elektroforézisen alapuló elemzések eredményei a kutatók számára világszerte hozzáférhetővé válnak.
Summary
Structural determination of glycans is a difficult task mainly due to the large diversity of sugar structures found in nature, the complex biosynthetic path variations and the physical and chemical similarities of oligosaccharides. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is an excellent tool to analyze fluorescently labeled glycans including the determination of positional and linkage isomers. Albeit, CE-LIF offers superior efficiency and resolving power, as well as very short separation times compared to chromatography techniques, there is no currently existing database to search structures assigned relative to migration times and glucose unit values (GU). This calls for the development of a comprehensive CE database of GU values to help in depth structural assignment of glycans. To fill this gap, we propose to build such a CE sugar unit database for a large number of N-linked glycans using three different homooligosaccharide ladders: (Glcα1,4)n, (Glcβ1,4)n and (GlcNAc)n. It is expected that the majority of the detected glycans can be structurally assigned based on their GU value information, but additional exoglycosidase treatment and mass spectrometry analysis information will be obtained should co-elution of different structures occur. The glycan database will be publicly accessible through a user-friendly web-based interface and thus bring CE separation based glycan analysis close to the researcher community.




 

Final report

  
Results in Hungarian
Az OTKA K-81839 sz. pályázat támogatásával felállítottunk egy glükózegység (GU) adatbázist, melyben kapilláris elektroforézis vizsgálatok során kapott elválasztások normalizált adatai nyilvánosan elérhetők. Nagyszámú glikánt analizáltunk lézer indukált detektálással kapcsolt kapilláris elektroforézissel (CE-LIF). Az adatbázisban a CE migrációs pozíciókat normalizált GU-értékekkel fejeztük ki. A glikánok szerkezetének megállapítása céljából a szénhidrátokat kombinált exoglikozidáz-emésztéseknek vetettük alá, melyet CE-LIF analízis követett. A többszörösen szialilált glikánok szerkezetének alaposabb megismerése érdekében előzetes gyenge anioncserés kromatográfiás frakcionálást alkalmaztunk. Az adatbázis a következő adatokat tartalmazza: (i) a monoszacharid szekvenciákat és kötőhelyeket bemutató grafikus és szimbolikus szerkezeti ábrák, (ii) GU értékben kifejezett CE migrációs idők, szórásokkal, (iii) információk az exoglikozidáz-emésztés után kapott termékekről és azok szerkezetéről, (iv), átfogó irodalomjegyzék. A projektet kiterjesztettük egy, a humán plazmaproteomot reprezentáló Analitkönyvtár előállításával, melyet kromatográfiás és kicsapási technikák kombinálásával hoztunk létre monoklonális antitest-proteomikai vizsgálatokhoz, és melynek segítségével szignifikáns glikozilációs különbségeket jellemezhetünk. A szénhidrát-szerkezeti adatbázist a egy felhasználóbarát webfelületen tettük elérhetővé. (http://glycobase.nibrt.ie/glycobase/show_nibrt.action).
Results in English
With the support of the OTKA K-81839 grant, we established a publicly available glucose unit (GU) database for capillary electrophoresis (CE) profile allocation. A large number of glycans was analyzed by CE with laser induced fluorescence detection (LIF) establishing a relational database containing migration positions expressed as GU values. Structural elucidation of glycans by consecutive enzymatic digestion of carbohydrates using exoglycosidase combinations, followed by CE-LIF analysis was also implemented. In addition, offline weak anion exchange chromatography fractionation was used for deeper structural studies of highly sialylated glycans. Database entries include (i) a graphical and symbolical representation of the structure depicting monosaccharide sequence and linkages, (ii) a display of CE migration time expressed as GU values with the corresponding standard deviations, (iii) information containing exoglycosidase digest product and mass values, and (iv) an extensive literature reference list. An extension of the project used combined chromatographic and precipitation techniques to generate a large set of fractions representing the human plasma proteome (Analyte Library), with the goal to use in conjunction with mAb proteomics to characterize significant glycosylation differences. The carbohydrate structure database was posted online through a user friendly web-based interface serving the research community (http://glycobase.nibrt.ie/glycobase/show_nibrt.action).
Full text https://www.otka-palyazat.hu/download.php?type=zarobeszamolo&projektid=81839
Decision
Yes





 

List of publications

  
Guttman A (Editor): Biomarker discovery and related topics, Electrophoresis, 32(15) 1915, 2011
Guttman A (Editor): Analytical Glycomics, Electrophoresis vol 34, #16: 2283, 2013
Szabo Z, Guttman A, Bones J, Shand RL, Meh D, Karger BL: Ultrasensitive capillary electrophoretic analysis of potentially immunogenic carbohydrate residues in biologics: 1. Galactose-α-1,3-Galactose Containing Structures, Molecular Pharmaceutics 9: 1612-1619., 2012
Szabo Z, Bones J, Guttman A, Glick J, Karger BL: Sialic Acid Speciation using Capillary Electrophoresis: Optimization of Analyte Derivatization and Separation, Analytical Chemistry 84: 7638-7642., 2012
Vanderschaeghe D, Guttman A, Callewaert N: High-throughput profiling of the serum N-glycome on capillary electrophoresis microfluidics systems, Methods Mol Biol. 919: 87-96., 2013
S.Tummala, M.Titus, L.Wilson, C.Wang, C.Ciatto, D.Foster, Z.Szabo, A.Guttman, C.Li, B. Bettencourt, M.Jayaraman, J.Deroot, G.Thill, D.Kocisko, S.Pollard, K.Charisse, S.Kuchimanchi, G.Hinkle, S.Milstein, R.Myers, S.Wu, B.L.Karger, A.Rossomando: RNAi-mediated metabolic engineering: Modulation of fucose content on an anti-CD20 monoclonal antibody using exogenously added siRNA, Biotech. Prog., 29: 415-424., 2013
Guttman A: Capillary electrophoresis in the N-glycosylation analysis of biopharmaceuticals, Trends in Analytical Chemistry 48: 132–143., 2013
Wang Y, Santos M, Guttman A: Comparative core fucosylation analysis of some major therapeutic antibody N-glycans by direct infusion electrospray ionization mass spectrometry and capillary electrophoresis – laser induced fluorescence detection, Journal of Separation Science. In press, 2013
Váradi C, Lew C, Dewald H, Thorn J, Guttman A: Magnetic bead based novel sample preparation method for automated N-glycosylation analysis of biotherapeutics by capillary electrophoresis, HPLC 2013, Amsterdam, The Netherlands, June 16-20, 2013
Guttman A, Szekrényes Á, Mittermayr S: Glucose Unit (GU) database for N-glycosylation identification of biopharmaceuticals by capillary electrophoresis, HPLC 2013, Amsterdam, The Netherlands, June 16-20, 2013
Krenkova J, Szekrenyes Á, Keresztessy Z, Foret F: Oriented Immobilization of PNGase F on a Monolithic Support for Glycoprotein Analysis, HPLC 2013, Amsterdam, The Netherlands, June 16-20, 2013
M.Guergova-Kuras, I.Kurucz, W.Hempel, N.Tardieu, J.Kadas, C.Malderez-Bloes, A.Jullien, Y.Kieffer, M.Hincapie,A.Guttman, E.Csanky, S.Cseh, B.L.Karger. L.Takacs: Discovery of lung cancer biomarkers by profiling the plasma proteome with nascent monoclonal antibody libraries, Molecular and Cellular Proteomics 10 (2011) M111.010298-1-14., 2011
Szabo Z, Guttman A, Bones J, Karger BL: Rapid High Resolution Characterization of Functionally Important Monoclonal Antibody N-Glycans by Capillary Electrophoresis, Analytical Chemistry 83:5329-5336., 2011
Szalma J, Boddi K, Lempel E, Fsieroslawska A, Szabo Z, Harfo R, Olasz L, Takatsy A, Guttman A: Proteomic and Scanning Electron Microscopic Analysis of Submandibular Sialoliths, Clinical Oral Investigations 17(7):1709-17, 2013
Guttman A, Szekrenyes A, Varadi C, Kerekgyarto M: New advances in capillary electrophoresis of therapeutic antibodies, CECE 2012, Brno, Czech Republic, Nov 1-2, 2012
Guttman A: Analytical Glycomics: New Tools for the Analysis of Biotherapeutics, CE User Meeting, Amgen, Thousand Oaks, CA, Nov 19, 2012
Guttman A, Mittermayr S, Varadi C, Szekrenyes A: Recent progress in analytical glycomics, LACE 2012, Buenos Aires, Argentina, Dec 2-4, 2012
Guttman A: The role of capillary electrophoresis in analytical glycomics, University of Pardubice, CZ, March 25, 2013
Guttman A: The analytical challenges of glycomics, Masaryk University, Brno, CZ, March 28, 2013
Guttman A: Analytical glycomics and glycol-informatics: new tools for the analysis of biotherapeutics, Biogen-Idec, Cambridge, MA, April 25, 2013
Guttman A, Szekrenyes A, Mittermayr S: Glucose Unit (GU) database for N-glycosylation identification of biopharmaceuticals by capillary electrophoresis, Balaton Conference, Siofok, HU, Sept 4-6, 2013
Guttman A: Analytical Glycomics: past, present and future, APRS 2013, Innsbruck, AT, Sept 23-25, 2013
Mittermayr S, Bones J, Doherty M, Guttman A, Rudd PM: Rapid and confident IgG N-glycan structural elucidation, J Proteome Res 10: 3820-3829., 2011
Kovács A, Sperling E, Lázár J, Balogh A, Kádas J, Szekrényes Á, Takács L, Kurucz I, Guttman A: Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery, Electrophoresis 32: 1916-1925., 2011
Mittermayr S, Bones J, Szabó Z, Rudd PM, Karger BL, Guttman A: Capillary electrophoresis based analytical and structural prediction tools for glycan analysis, 7th International Interdisciplinary Meeting on Bioanalysis, 2010
Guttman A: Structural prediction of N-linked glycans by capillary electrophoresis and capillary electrochromatography, 16th Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical And Industrial Applications Of Capillary Electrophoresis And Microchip Technology, 2010
Garrido-Medina R, Puerta A, Rivera-Monroy Z, de Frutos M, Guttman A, Diez-Masa JC: Analysis of alpha-1-acid glycoprotein isoforms using CE-LIF with fluorescent thiol derivatization, Electrophoresis 33: 1113-1119., 2012
Mesko B, Poliska Sz, Szekanecz Z, Szamosi Sz, Podani J, Varadi Cs, Guttman A, Nagy L: Peripheral Blood Gene Expression and IgG Glycosylation Profiles as Markers of Tocilizumab Treatment in Rheumatoid Arthritis, The Journal of Rheumatology 39: 916-928., 2012
Mittermayr S, Guttman A: Influence of molecular configuration and conformation on the electromigration of oligosaccharides in narrow bore capillaries, Electrophoresis 33: 1000-1007., 2012
Guttman A: Bioanalytical Tools for the Characterization of Biologics and Biosimilars, LC.GC Magazine 30 412-421., 2012
Kerékgyártó M, Kerekes T, Tsai E, Amirkhanian VD, Guttman A: Light emitting diode induced fluorescence (LedIF) detection design for a pen-shape cartridge based single capillary electrophoresis system, Electrophoresis 33: 2752-2758., 2012
Szekrényes Á, Roth U, Székely A, Kurucz I, Kowalewski K, Guttman A: High throughput monoclonal antibody analysis by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling, Anal.Bioanal.Chem. 405: 1485-1494., 2012
Guttman A, Szabó Z, Karger BL: High pressure mediated sample preparation for capillary electrophoresis analysis of N-linked glycans, 49th EHPRG Conference, 2011
Guttman A: Translational Glycomics, Institute of Analytical Chemistry, Brno, 2012
Guttman A: Analytical Glycomics, Proteomics Workshop, Aviero, Portugal, 2012
Guttman A, Szabo Z, Bones J, Karger BL, Shan R, Meh D: Ultrasensitive capillary electrophoresis analysis of potentially immunogenic carbohydrate residues in biologics, Absztrakt #L-02-08, HPLC 2012, Anaheim, CA, 2012
Guttman A, Váradi Cs, Meskó B, Nagy L: Capillary electrophoresis analysis of the altered glycosylation of immunoglobulins in autoimmune diseases, CECE 2011, Brno, 2011
Guttman A, Szekrenyes A, Szekely A, Kurucz I: Large scale heterogeneity analysis of monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling, LACE 2011, Miami, 2011
Guttman A, Mittermayr S, Bones J, Rudd PM: High fidelity structural elucidation of IgG N-glycans by orthogonal pressure and electric field mediated separations, Absztrakt #KN9, MSB 2012, Geneva, Switzerland, 2012
Guttman A, Szabo Z, Karger BL, Chapman J, Kimzey M, Haxo T, Wegstein J: Rapid Sample Preparation of Biologics to Support High-throughput/High-resolution Glycan Analysis by Capillary Electrophoresis, Absztrakt #P-117, CE-PHARM 2011, Amelia Island, FL, 2011
Kimzey M, Pourkaveh S, Mast S, Sun B, Minalla A, Haxo T, Nishida C, Karger BL, Guttman A, Szabo Z, Chapman J, Wegstein J: A High-Throughput Platform for Preparation of APTS-Labeled N-Glycans: Improving the Accuracy, Reproducibility and Time-to-Results of N-Glycan Profiling, CE-PHARM 2011, Amelia Island, FL, 2011
Szabo Z, Karger BL, Chapman J, Kimzey M, Haxo T, Wegstein J, Guttman A: Automated Sample Preparation of Biologics for High-throughput/High-resolution Glycan Analysis, PTM 2011, Berlin, Germany, 2011
Guttman A, Szabo Z, Karger BL, Chapman J, Kimzey M, Haxo T, Wegstein J: Towards the Automated Sample Preparation of Biotherapeutics for High-throughput/High-resolution Glycan Analysis by CE, Bioprocess International, Long Beach, CA, 2011
Guttman A, Szabo Z, Karger BL, Chapman J, Kimzey M, Haxo T, Wegstein J: Rapid Sample Preparation of Biologics to Support High-throughput/High-resolution Glycan Analysis by Capillary Electrophoresis, Annual Conference of the Society for Glycobiology, Seattle, WA, 2011
Szekrenyes A, Kimzey M, Pourkaveh S, Mast S, Sun B, Minalla A, Haxo T, Nishida C, Wegstein J, Szabo Z, Karger BL, Chapman J, Guttman A: A High-Throughput Platform for Preparation of APTS-Labeled N-Glycans: Improving the Accuracy, Reproducibility and Time-to-Results of N-Glycan Profiling, Absztrakt #36, CECE 2011, Brno, 2011
Kovács A, Patai Z, Sperling E, Lázár J, Balogh A, Kádas J, Szekrényes Á, Takács L, Kurucz I, Guttman A: Biomarker screening of the human plasma proteome with disease specific monoclonal antibodies, Absztrakt #16, CECE 2011, Brno, 2011
Kerékgyártó M, Bacskai I, Guttman A: Rapid Analysis of Genomic DNA Samples by a Novel Capillary Gel Electrophoresis Platform, Absztrakt #15, CECE 2011, Brno, 2011
Váradi Cs, Rezsu N, Meskó B, Guttman A: Analysis of Altered IgG Galactosylation in Autoimmune Diseases, Absztrakt #37, CECE 2011, Brno, 2011
Guttman A, Szabo Z, Karger BL, Wegstein J, Nishida C, Chapman J, Dewald H, Mittermayr S, Bones J, Doherty M, Rudd P: Rapid Sample Preparation of Biologics to Support High-throughput and High-resolution Glycan Analysis by Capillary Electrophoresis, WCBP 2012, San Francisco, 2012
Kimzey M, Pourkaveh S, Mast S, Sun B, Minalla A, Haxo T, Nishida C, Wegstein J, Guttman A, Szekrenyes A, Szabo Z, Karger BL, Chapman J, Dewald H: A High-Throughput Platform for Preparation of APTS-Labeled N-Glycans: Improving the Accuracy, Reproducibility and Time-to-Results of N-Glycan Profiling, WCBP, San Francisco, 2012
Váradi Cs, Meskó B, Guttman A: CE-LIF based evaluation of treatment efficacy in autoimmune diseases: deciphering the relationship between IgG N-glycosylation and mRNA expression profile, Absztrakt #P355, MSB 2012, Genf, 2012
Mittermayr S, Bones J, Guttman A, Rudd P: Identification of Cancer Associated Alterations in Serum N-Glycosylation by Capillary Electrophoresis, Absztrakt #P385, MSB 2012, Genf, 2012
Kovács A, Patai Z, Kádas J, Takács L, Kurucz I, Guttman A: Monoclonal antibody proteomics: antigen identification by mini dot-blot array screening, Absztrakt #P812, MSB 2012, Geneva, 2012
Kerékgyártó M, Bacskai I, Guttman A: Large Scale Genomic DNA Analysis by Capillary Electrophoresis, Absztrakt #P403, MSB 2012, Geneva, 2012
Patai Z, Kovács A, Kurucz I, Takács L, Guttman A: Monoclonal antibody proteomics based characterization of a human plasma proteome library, Absztrakt # P-415-Tue, HPLC 2012, Anaheim, 2012
Kovács A, Guttman A: Medicinal Chemistry Meets Proteomics: Fractionation of the Human Plasma Proteome, Current Medicinal Chemistry 20 (2013) 483-490., 2013
Kerékgyártó M, Németh N, Kerekes T, Rónai Zs, Guttman A: Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis, J.Chrom. A. 1286 (2013) 229-234., 2013
Váradi Cs, Mittermayr S, Szekrényes Á, Kádas J, Takács L, Kurucz I, Guttman A: Analysis of haptoglobin N-glycome alterations in inflammatory and malignant lung diseases by capillary electrophoresis, Electrophoresis, In Press, 2013
Mittermayr S, Bones J, Guttman A: Unraveling the Glyco-Puzzle: Glycan Structure Identification by Capillary Electrophoresis, Anal.Chem. 85 (2013) 4228-4238, 2013
Kerékgyártó M, Fekete A, Szurmai Z, Kerékgyártó J, Kurucz I, Guttman A: Neoglycoproteins for carbohydrate specific antibody generation 1: Synthesis, analysis and polyclonal antibody response, Electrophoresis. In press, 2013
Németh N, Kerékgyártó M, Sasvári-Székely M, Rónai Z, Guttman A: Rapid identification of human SNAP-25 transcript variants by a miniaturized capillary electrophoresis system, Electrophoresis In Press, 2013
Kovács A, Patai Z, Guttman A, Kádas J, Takács L, Kurucz I: Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery 2: Antigen identification by dot-blot array screening, Electrophoresis. In press, 2013
Guttman A: Analysis of potentially immunogenic carbohydrate residues in biologics, ITP 2012, Baltimore, MD, Sept 30-Oct 3, 2012
Guttman A: Glycosylation primer, 6th CEEPC, Budapest, Hungary, Oct 14-17, 2012



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