English version
 

Proiect PNCD II nr. 41001/2007

Titlu Identificarea si caracterizarea biomarkerilor cancerului de creier exprimati in ser si in lichidul cefalorahidian prin metode glicomice avansate bazate pe spectrometrie de masa
   
Acronim  BRAIN_CAN_GLICOMICS
   
Rezumatul proiectului
 Cancerele creierului sunt un grup de boli severe de obicei detectate prin metode de diagnosticare costisitoare, bazate pe imagistica, aplicate intr-un stadiu avansat al bolii si cu un prognostic rezervat. Cea mai practica alternativa este detectarea precoce a tumorilor intr-un stadiu in care mai pot fi rezectate si care trebuie sa se bazeze pe un screening de rutina si descoperire de biomarkeri cu mult inainte de aparitia simptomelor. De aceea, in prezent se impune elaborarea unor tehnici si protocoale mai simple pentru a permite translatarea rezultatelor cercetarilor de laborator in aplicatii clinice viabile. In acest context se propune aici un proiect interdisciplinar in cadrul PNCD II folosind tehnici avansate de spectrometrie de masa, nanotehnologii si medicina focalizate pe glicomica gangliozidelor si glicozaminoglicanilor exprimati in fluidele corpului bolnavilor de cancer la creier. In acest proiect vor fi dezvoltate metode avansate de MS de o sensibilitate foarte mare, automatizate, bazate pe chip si proceduri de screening analitic de inalta performanta pentru a stabili schimbarile in tiparul expresiei gangliozidelor si glicozaminoglicanilor ca biomarkeri in diagnosticul precoce, evaluarea si descoperirea tumorilor creierului. Analiza de inalta sensibilitate prin spectrometrie de masa va include si identificarea si caracterizarea biomarkerilor prin fragmentare MS in tandem prin stagii multiple. Vor fi colectate, comparate si procesate date despre compozitia specifica a gangliozidelor si glicozaminoglicanilor, epimerizare, secventiere, tipare de sulfatare si diferite alte modificari structurale si de compozitie ca si abundenta in ser si in lichidul cefalorahidian al pacientilor diagnosticati cu cancer in comparatie cu subiecti sanatosi. Principala conditie preliminara a acestui studiu o reprezinta integrarea stiintelor vietii cu spectrometria de masa si cu tehnologia microfluidica si va fi indeplinita de un consortiu ce intruneste diverse expertize in domenii de cercetare complementare. Metodele noi de descoperire a biomarkerilor dezvoltate in acest proiect pentru colectarea de date si sortarea informatiilor esentiale vor forma o platforma eficienta, capabila sa influenteze directiile de cercetare ale cancerului. Este de asteptat ca acest sistem integrativ va gasi aplicabilitate generala ca metodologie rapida, precisa si neinvaziva capabila de diagnosticarea precoce a cancerului de creier, evaluarea agresivitatii sale si a tratamentului ulterior in scopul imbunatatirii sistemului sanitar in U.E. si inafara acesteia.
   
Obiectivele generale
Obiectivul proiectului este de a dezvolta noi metode glicomice si glicoproteomice bazate pe spectrometria de masa de inalta performanta in combinatie cu metode de infuzie robotizata si tehnologie chip/microfluidica in scopul descoperirii biomarkerilor cancerului la creier intr-un stadiu incipient al bolii si pentru monitorizarea progresiei tumorii. Metodele ce urmeaza a fi dezvoltate se bazeaza pe tehnologii inovatoare de microfluidice/MS care vor fi concepute, testate si pentru prima data aplicate analizei glicoconjugatilor in fluidele organismului precum sange si lichid cefalorahidian.
   
Institutia coordonatoare Universitatea de Medicina si Farmacie Carol Davila din Bucuresti
   
Componenta consortiului Universitatea de Medicina si Farmacie Carol Davila din Bucuresti

Institutul National de Cercetare-Dezvoltare pentru Electrochimie si Materie Condensata Timisoara

Universitatea Aurel Vlaicu din Arad

Universitatea de Medicina si Farmacie Victor Babes din Timisoara
   
Autoritatea contractanta  Centrul National de Management Programe
   
Durata proiectului;  36 luni
   
Descrierea etapelor
proiectului
Etapa I / 2007: Studiul progreselor actuale in diagnosticarea cancerului la creier prin metode de imagistica (15.12.2007)

Etapa II/2008: Evaluarea comparativa a tendintelor actuale in cercetarea cancerului la creier prin metode de "omics". Identificarea/selectarea cazurilor (pacienti si donori sanatosi). (30.10.2008)

Etapa III/2009: Prelevarea si pregatirea probelor din ser si fluid cefalorahidian (LCR). Izolarea si purificarea amestecurilor complexe de glicozaminoglicani (GAG) din LCR (30.05.2009)
Etapa IV/2009: Izolarea si purificarea amestecurilor complexe de gangliozide (GG) din ser si LCR (30.11.2009)

Etapa V/2010: Screening-ul si fragmentarea GAG si GG prin chip-nanoelectrospray QTOF MS, HCT MSn si HPLC HCT MS/MS (14.09.2010)
   
Datele de contact ale directorului de proiect

Prof. univ. Leon Zagrean
email: lzagrean @ univermed-cdgm.ro
Bd. Eroii Sanitari, nr. 8, 050474 Bucuresti

   
 Rezultate

Articole publicate ISI

1. Glycosylation analysis of glycoproteins and proteoglycans using capillary electrophoresis-mass spectrometry strategies
Sabine Amon, Alina D. Zamfir, Andreas Rizzi
Electrophoresis 2008, 29, 2485–2507, DOI 10.1002/elps.200800105 (ISI)

2. High-Throughput Analysis of Gangliosides in Defined Regions of Fetal Brain by Fully Automated Chip-Based Nanoelectrospray Ionization Multistage Mass Spectrometry.
Alina Serb, Catalin Schiopu, Corina Flangea, Željka Vukelic, Eugen Sisu, Nicolae Dinca, Leon Zagrean and Alina D. Zamfir
Eur. J. Mass Spectrom. 15, 541-553, 2009 (ISI)

3. Synthesis and structural characterization of amino-functionalized polysaccharides
I. Sisu, V. Udrescu, C. Flangea, S. Tudor, N. Dinca, L. Rusnac, A.D. Zamfir, E.Sisu
Cent. Eur. J. Chem. 7, 66-73, 2009 (ISI)

4. Fully automated chip-based negative mode nanoelectrospray mass spectrometry of
fructooligosaccharides produced by heterologously expressed levansucrase from Pseudomonas syringae pv. tomato DC3000
T.Visnapuu, A. D. Zamfir, C. Mosoarca, M. D. Stanescu, T.Alamäe
Rapid Commun. Mass Spectrom. 23, 1337–1346, 2009 (ISI, IF=3.05)

5. Analysis of novel over- and undersulfated glycosaminoglycan sequences by enzyme
cleavage and multiple stage mass spectrometry
A.D. Zamfir, C. Flangea, A. Serb, E.Sisu, N. Dinca, P. Bruckner, D. G. Seidler
Proteomics 9, 1-10, 2009 (ISI, IF=5.87)

6. Top-down glycolipidomics: fragmentation analysis of ganglioside oligosaccharide core and
ceramide moiety by chip-nanoelectrospray collision-induced dissociation MS2-MS6
A. Serb, C. Schiopu, C. Flangea, E. Sisu, A.D. Zamfir. J. Mass Spectrom. 2009 (ISI, IF=3.21)

7. Discrimination of GalNAc (4S/6S) sulfation sites in chondroitin sulfate disaccharides by
chip-based nano-electrospray multistage mass spectrometry
C. Flangea, A. Serb, C. Schiopu, S. Tudor, E. Sisu, D. G. Seidler, A. D. Zamfir
Cent. Eur. J. Chem. 2009 (ISI)

8. Qualitative and quantitative analysis of gallic acid in Alchemilla vulgaris, Allium ursinum, Acorus calamus and Solidago virga-aurea by chip-electrospray ionization mass spectrometry and high performance liquid chromatography
Dumitru Condrat, Cristina Mosoarca, Alina D. Zamfir, Florin Crisan, Maria R. Szabo and Alfa X. Lupea
Central European Journal of Chemistry, vol. 3, no. 3, June 2010 (ISI)


Comunicari stiintifice

1.Screening si Fragmentare Comparativa a Gangliozidelor din Creierul Fetal Uman prin Metode Avansate de Spectrometrie de Masa
Alina F. Serb, Eugen Sisu, Corina Flangea, Alina D. Zamfir, Leon Zagrean,
Conferinta Diaspora in Cercetarea Stiintifica Romaneasca, Bucuresti, 17-19 septembrie 2008

2.Sistem Informatic de Operare pentru Interpretarea Spectrelor de Masa a Gangliozidelor din Creierul Uman
Catalin Schiopu, Eugen Sisu, Sorin Nadaban, Carmen Fifor, Alina Zamfir, Conferinta Diaspora in Cercetarea Stiintifica Romaneasca, Bucuresti, 17-19 septembrie 2008

3. Determination and structural characterization of anencephaly-associated gangliosides by advanced mass spectrometry based on chip electrospray ionization and multistage sequencing. A. D., Zamfir, C. Mosoarca, C.Flangea, Z. Vukelic., E. Sisu
6th Forum of European Neuroscience, 12-16 July 2008 Geneva, Switzerland; FENS Abstr., vol.4, 083.29, 2008

4. Comparative assay on ganglioside differential expression in defined regions of uman brain by modern mass spectrometric methods.
A. F., Serb, C. Flangea, Z.Vukelic, E.Sisu , L.Zagrean, A. Zamfir
6th Forum of European Neuroscience, 12-16 July 2008 Geneva, Switzerland; FENS Abstr., vol.4, 075.11, 2008

5. Gangliozidele prezinta o inalta specificitate filogenetica in creierul fetal uman, demonstrata prin investigatii de spectrometrie de masa nanoESI bazate pe chip
A.F.Serb, E.Sisu, C.Schiopu, A.D. Zamfir, L.Zagrean
Al IV-lea Simpozion al Societatii Nationale de Neurostiinte, 30-31 mai 2008, Bucuresti, Romania;Abstr.Vol. (ISBN: 978-973-708-323-4), p.42

6. Investigation of glycosaminoglycan sulfation pattern by high performance chip electrospray multistage mass spectrometry
C.Flangea, A.F. Serb, S.Tudor, I.B.Pancan, E.Sisu, D.G.Seidler, A.D.Zamfir
International Symposium Research and Education in Innovation Era, 2nd Edition, November 20th - 21st , 2008

7. Computer software for the interpretation brain ganglioside mass spectra
C.Schiopu, E.Sisu, Ž.Vukelic, N.Dinca, S.Nadaban, C.Fifor, A.D.Zamfir
International Symposium Research and Education in Innovation Era, 2nd Edition, November 20th - 21st, 2008

8. Profiling of gangliosides expressed in human hemangioma by fully-automated chip-based nanoelectrospray tandem mass spectrometry
A.Serb, C.Schiopu, C.Flangea, E.Sisu, S.Tudor, I.B.Pancan,Ž. Vukelic, A.D. Zamfir
International Symposium Research and Education in Innovation Era, 2nd Edition, November 20th - 21st, 2008

9. Potentials of a fully automated chip-based nanoelectrospray ionization high capacity trap mass spectrometry in neuroglycolipidomics: characterization of ganglioside composition
specificity in selected fetal human brain regions
Ž.Vukelic, D. Marincic, A.Serb, A. D. Zamfir
Invited lecture: Austrian Mass Spectrometry Forum, Feb. 17-19, 2009, Vienna, Austria

10. Novel saccharide sequence analysis of glycosaminoglycans by enzyme cleavage and multistage MS: unusual exuronic acid sulfation in decorin
C. Flangea, C. Schiopu, E. Sisu, N. Dinca, D. G. Seidler, A. D. Zamfir
Oral presentation: Austrian Mass Spectrometry Forum, Feb. 17-19, 2009, Vienna, Austria

11. A new Mass Spectrometry Society – Perspectives and plans
A.D. Zamfir
Invited lecture: Anniversary Conference, 20 years of Analytical Chemistry in Konstanz,
March 5-7, 2009, Konstanz, Germany

12. Chip-based nanoelectrospray multistage mass spectrometry in neuroglycolipidomics: high-throughput analysis of gangliosides in defined regions of fetal brain
A.F. Serb, C. Schiopu, D. Marincic, Ž. Vukelic, A.D. Zamfir
Oral presentation: 42nd German Society for Mass Spectrometry Conference, Konstanz,
Germany, March 8-11, 2009

13. Investigation of novel under- and over-sulfated regions in decorin glycosaminoglycans by electrospray ionization collision-induced dissociation multistage mass spectrometry
C. Flangea, E. Sisu, N. Dinca, D.G. Seidler, A. D. Zamfir
Poster presentation: 42nd German Society for Mass Spectrometry Conference, Konstanz,
Germany, March 8-11, 2009

14. A novel procedure for derivatization of long chain polysaccharides to enhance chip-based nanoelectrospray tandem mass spectrometric analysis
C. Flangea, I.Sisu, N. Dinca, E. Sisu, A. D. Zamfir
Poster presentation: 42nd German Society for Mass Spectrometry Conference, Konstanz,
Germany, March 8-11, 2009

15. Chip-Based nanoelectrospray high capacity ion trap mass spectrometry of gangliosides from precentral vs. postcentral gyrus of fetal human brain
D.Marincic, Ž.Vukelic, C. Schiopu, M. Kos, A. D. Zamfir
Poster presentation: 42nd German Society for Mass Spectrometry Conference, Konstanz,
Germany, March 8-11, 2009

16. Screening and sequencing of fructooligosaccharides produced by heterologously expressed levansucrase from Pseudomonas syringae pv. tomato DC3000 by fully automated chip based mass spectrometry
C. Mosoarca, T. Visnapuu, T. Alamäe, A. D. Zamfir
Poster presentation: 42nd German Society for Mass Spectrometry Conference, Konstanz,
Germany, March 8-11, 2009

17. High-throughput mass spectrometric determination of ganglioside expression and structure in human fetal cerebellum
R. M. Ghiulai, C. Mosoarca, C. Novaconi, D. Marincic, Ž. Vukelic, A. D. Zamfir
Poster presentation: 42nd German Society for Mass Spectrometry Conference, Konstanz,
Germany, March 8-11, 2009

18. Chip-based mass spectrometry for glycomics and glycoproteomics
A. D. Zamfir
Invited lecture: University of Konstanz, May 13, 2009

19. Analysis of brain chondroitin/dermatan sulfate glycosaminoglycans by fully automated chip-based nanoelectrospray multistage mass spectrometry
C. Flangea, E. Sisu, D. G. Seidler, A. D. Zamfir
Poster presentation: 57th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, May 31-June 6, 2009, Philadelphia, USA

20. Compositional and structural analysis of gangliosides in human cerebrospinal fluid by chip-based nanoelectrospray ionization tandem mass spectrometry
A. Serb, C. Schiopu, D. Marincic, Ž. Vukelic, A. D. Zamfir
Poster presentation: 57th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, May 31-June 6, 2009, Philadelphia, USA

21. Combining fully-automated chip-nanoelectrospray ion trap mass spectrometry and GanglioSoft 1.2 computer software for identification of human hemangioma gangliosides
C.Schiopu, A. Serb, E. Sisu, Ž. Vukelic, A. D. Zamfir
Poster presentation: 57th American Society for Mass Spectrometry Conference on Mass
Spectrometry and Allied Topics, May 31-June 6, 2009, Philadelphia, USA