Development of microfluidics-mass spectrometry for the analysis of ganglioside structural and functional interactions

Development of microfluidics-mass spectrometry for the analysis of ganglioside structural and functional interactions

CONTRACT 48 from 12/07/2017

PN-III-P4-ID-PCE-2016-0073

Proiect Director CS I Dr. Fiz. Alina-Diana Zamfir

Abstract

Gangliosides (GGs), sialylated glycosphingolipids, are plasma membrane components and valuable markers of central nervous system pathologies. The interest in the study of GG interactions with different molecules was stimulated by findings indicating that the formed complexes play significant biological roles. In the last years, our group introduced microfluidics-mass spectrometry (MS) in ganglioside research. This technique exhibits a number of advantages, which make it highly suitable for detection and characterization of the complexes formed by GGs with peptides and proteins. In view of this state-of-the-art, the objective of the present proposal is the development of high performance microfluidics (chip-nanoelectrospray ionization, nanoESI)-MS for determination of normal human brain and brain tumor ganglioside interactions with neuropeptides and proteins. Structured in 4 workpackages (WP), the project targets the implementation of chip-nanoESI MS for the study of neuropeptide-GG interactions in WP1, the method optimization for protein-GG interactions in WP2 and an assay for following neuropeptide and protein interactions with brain tumor-GG in WP3. The interaction assays will also include the detailed characterization of the formed complexes in terms of binding site(s) identification and determination of the bound GG species structure by top-down complex fragmentation in multistage MS (MSn) experiments. Chip-based nanoESI will be performed on a NanoMate robot in direct coupling with two advanced MS instruments: a hybrid quadrupole time-of-flight and a high capacity ion trap MS. WP4 is dedicated to result dissemination through publications in highly ranked international journals and presentations at prominent conferences. It is expected that this novel analytical platform will progress far beyond the project lifetime, into a routine method of broader applicability for the analysis of interactions between various classes of biomolecules.

Estimated results

The estimated results are:

i) a novel analytical platform and approach based on microfluidics-MS optimized for detection and structural analysis of ganglioside-neuropeptide and ganglioside-protein noncovalent complexes;

ii) validation of the developed protocols for the interactions of multicomponent ganglioside extracts originating from human brain and cerebral tumors;

iii) identification of ganglioside-peptide/protein interactions potentially relevant to various brain processes/pathologies;

iv) discovery of novel ganglioside species in biological mixtures based on their protein binding affinity.

Published articles

1) I.I. Cozma, M. Sarbu, C. Ilie, A.D. Zamfir, Structural analysis by electrospray ionization mass spectrometry of GT1 ganglioside fraction isolated from fetal brain, Journal of Carbohydrate Chemistry (2017), 36:247-264. Impact factor 0.629

2) M. Sarbu, A.D. Zamfir, Modern separation techniques coupled to high performance mass spectrometry for glycolipid analysis, Electrophoresis (2018), 39(9-10):1155-1170. Impact factor 2.569

3) A.C.Robu *, L.Popescu *, D.G. Seidler, A.D. Zamfir, Chip-based high resolution tandem mass spectrometric determination of fibroblast growth factor-chondroitin sulfate disaccharides noncovalent interaction, Journal of Mass Spectrometry (2018), 53(17):624-634 (*equal contribution). Impact factor 2.112.

4) M. Sarbu, Z. Vukelic, D.E. Clemmer, A.D. Zamfir, Ion mobility mass spectrometry provides novel insights into the expression and structure of gangliosides in normal adult human hippocampus, Analyst (2018), DOI:10.1039/C8AN01118D. Impact factor 3.864.

5) M. Sarbu, Z. Vukelic, D. E. Clemmer, A.D. Zamfir, Electrospray ionization ion mobility mass spectrometry provides novel insights into the pattern and activity of fetal hippocampus gangliosides, Biochimie 139: 81e94 (2017). Impact factor 3.188

6) M. Sarbu, C. Cozma, A. D. Zamfir, Structure-to-Function Relationship of Carbohydrates in the Mechanism of Lysosomal Storage Disorders (LSDs), Current Organic Chemistry, 21, 1-12 (2017).Impact factor 2.193

7) M. Sarbu, R. Ica, A. Petrut, Z. Vukelic´, C.V.A. Munteanu, A.J. Petrescu, A.D. Zamfir, Gangliosidome of human anencephaly: a high resolution multistage mass spectrometry study, Biochimi, pii: S0300-9084(19)30164-6. doi: 10.1016/j.biochi.2019.05.017. Impact factor 3.362

8) Albulescu R., Petrescu A.J., Sarbu M., Grigore A., Ica R., Munteanu C.V.A., Albulescu A., Militaru I. V, Zamfir A.-D., Petrescu S., Tanase C., Mass spectrometry for cancer biomarker discovery, IntechOpen, 2019 DOI: 10.5772/intechopen.85609

9) Dehelean L.*, Sarbu M.*, Petrut A., Zamfir A.D., Trends in glycolipid biomarker discovery in neurodegenerative disorders by mass spectrometry, Adv. Exp. Med. Biol. Journal. 1140:703-729 (2019) * equal contribution Impact factor 2.126

10) Mirela Sarbu, Liana Dehelean, Cristian V.A. Munteanu, Raluca Ica, Andrei J. Petrescu, Alina D. Zamfir., Human caudate nucleus exhibits a highly complex ganglioside pattern as revealed by high resolution multistage Orbitrap MS Journal of Carbohydrate Chemistry 2019 38(9): 531-551 doi:10.1080/07328303.2019.1669632. Impact factor:0.828

11) Mirela Sarbu, Shannon Raab, Lucas Henderson, Dragana Fabris, Zeljka Vukelic´, David E. Clemmer, Alina D. Zamfir, Cerebrospinal fluid: profiling and fragmentation of gangliosides by ion mobility mass spectrometry, Biochimie 2019(under evaluation)

12) Raluca Ica#, Alina Petrut#, Cristian V.A. Munteanu, Mirela Sarbu, Zeljka Vukelic´, Ligia Petrica and Alina D. Zamfir (# Equal contribution), Orbitrap mass spectrometry for monitoring the ganglioside pattern in human cerebellum development and aging, Journal of Mass Spectrometry (Submitted).

Conference presentations

[1]. Profiling and structural characterization by mass spectrometry of region-specific gangliosides in brain M. Sarbu, Z. Vukelic´, D.E. Clemmer, A.D. Zamfir 23rd International Symposium on Analytical and Environmental Problems, 9-10 October 2017, Szeged, Hungaria. ORAL PRESENTATION

[2]. High performance mass spectrometry for advanced interactomics studies A.C. Robu, L. Popescu, D.G. Seidler, A.D. Zamfir 23rd International Symposium on Analytical and Environmental Problems, 9-10 October 2017, Szeged, Hungaria. POSTER PRESENTATION

[3]. Modern mass spectrometric approaches for biomarker discovery A.D. Zamfir Walsh Seminar, 9-10 November 2017, Potsdam, N.Y., USA INVITED LECTURE

[4].Mass spectrometry of proteoglycans A.D. Zamfir Dept. of Chemistry & Biomolecular Science, 9 November 2017, Clarkson University, N.Y., USA INVITED LECTURE

[5]. Microfluidics-mass spectrometry for glycomics in biomedical research A.D. Zamfir, invited lecture Dept. of of Chemistry & Biomolecular Science, 10 November 2017, Clarkson University, N.Y., USA INVITED LECTURE

[6]. Chip-nanoESI QTOF MS of FGF2-CS noncovalent interaction A.C. Robu L. Popescu, M. Sarbu, D.G. Seidler, A.D. Zamfir 29th MassSpec Forum Vienna, 20-21 February 2018, Viena, Austria ORAL PRESENTATION

[7]. IMS MS of human fetal hippocampus: novel insights into the ganglioside domain M. Sarbu, Z. Vukelic, D.E. Clemmer, A.D. Zamfir 29th MassSpec Forum Vienna, 20-21 February 2018, Viena, Austria POSTER PRESENTATION

[8]. ESI MS/MS discovery of a novel brain GT1 ganglioside species I.I. Cozma, M. Sarbu, C. Ilie, A.D. Zamfir 29th MassSpec Forum Vienna, 20-21 February 2018, Viena, Austria POSTER PRESENTATION

[9]. High performance chip-nanoelectrospray tandem mass spectrometry characterization of the noncovalent FGF2 – CS complex Popescu L., A.D. Zamfir TIM 18 Physics Conference, 24-26 May, Timisoara, Romania ORAL PRESENTATION

[10]. Development of novel mass spectrometry approaches for the analysis of brain gangliosides and their structural and functional interactions, Popescu L., Sarbu M., Robu A.C., A.D. Zamfir 7th International Symposium Research and Education in Innovation Era, 17-20 May 2018, Arad, Romania. ORAL PRESENTATION

[11]. Fibroblast growth factor-glycosaminoglycan noncovalent interaction analysis by chip-nanoelectrospray mass spectrometry Robu AC, Popescu L, Seidler DG, A.D. Zamfir, ISREIE, Student Scientific Session, XVIth Edition, 10-12 May, 2018, Arad, Romania ORAL PRESENTATION

[12]. Determination of neuropeptides-human brain gangliosides noncovalent interactions by microfluidics tandem mass spectrometry Robu AC, Sarbu M, Popescu L, Vukelic Z, A.D. Zamfir Proceedings of the American Society for Mass Spectrometry Conference, 3-7 iunie 2018, San Diego, SUA

[13]. Ion mobility mass spectrometry of human brain hippocampus gangliosides Sarbu M, Vukelic Z, Clemmer D, A.D. Zamfir Proceedings of the American Society for Mass Spectrometry Conference, 3-7 June 2018, San Diego, SUA

[14]. Glycolipidomics in biomedical research by ion mobility mass spectrometry Mirela Sarbu, Adrian C. Robu, Alina D. Zamfir 15th International Conference on Nanosciences & Nanotechnologies (NN17), 3-6 July 2018, Salonic, Greece POSTER PRESENTATION

[15]. Protein-carbohydrate noncovalent interactions by microfluidics-mass spectrometry Adrian C. Robu, Mirela Sarbu, Alina D. Zamfir 15th International Conference on Nanosciences & Nanotechnologies (NN17), 3-6 July 2018, Salonic, Greece POSTER PRESENTATION

[16]. Ion mobility mass spectrometry for profiling and structural characterization of human hippocampus gangliosides Mirela Sarbu, Zeljka Vukelic´, David E. Clemmer, Alina Diana Zamfir 22nd International Mass Spectrometry Conference (IMSC) 2018, 26-31 August 2018, Florence, Italy) POSTER PRESENTATION

[17]. Glycolipidomics of human brain by combined modern mass spectrometry methods: chip nano ESI MS and ESI IMS MSn Mirela Sarbu, Zeljka Vukelic´, David E. Clemmer, Alina Diana Zamfir 22nd International Mass Spectrometry Conference (IMSC) 2018, 26-31 August 2018, Florence, Italy) POSTER PRESENTATION

[18]. Ion mobility mass spectrometry of gangliosides in human brain in health and disease Mirela Sarbu, Zeljka Vukelic´, David E. Clemmer, Alina Diana Zamfir 24rd International Symposium on Analytical and Environmental Problems, 8-9 October 2018, Szeged, Hungaria ORAL PRESENTATION

[19]. Advanced mass spectrometry methods for the determination of gangliosides structure and functional interactions Raluca Ica, Mirela Sarbu, Alina Diana Zamfir 24rd International Symposium on Analytical and Environmental Problems 8-9 October 2018, Szeged, Hungaria. POSTER PRESENTATION

[20]. Introducing ion mobility tandem mass spectrometry in glycoproteomics and glycolipidomics of human biopsies Mirela Sarbu, Zeljka Vukelic´, David E. Clemmer, Alina Diana Zamfir 12th Central and Eastern European Proteomic Conference (CEEPC), 24-26 October 2018, Bucharest, Romania. ORAL PRESENTATION.

[21]. Analyses of glycolipid-peptide and glycolipid-protein interactions by chip-based mass spectrometry Alina Diana Zamfir, Mirela Sarbu, Zeljka Vukelic´ 12th Central and Eastern European Proteomic Conference (CEEPC), 24-26 October 2018, Bucharest, Romania POSTER PRESENTATION

[22]. Protein-glycan noncovalent interactions: a fully automated chip-based mass spectrometry determination Mirela Sarbu, Zeljka Vukelic´, David E. Clemmer, Alina Diana Zamfir 12th Central and Eastern European Proteomic Conference (CEEPC), 12-14 November 2018, New Jersey, USA POSTER PRESENTATION

[23]. Ion mobility tandem mass spectrometry: a novel method in glycolipidomics of human brain. Applications to human hippocampus Mirela Sarbu, Zeljka Vukelic´, David E. Clemmer, Alina Diana Zamfir 12th Central and Eastern European Proteomic Conference (CEEPC), 12-14 November 2018, New Jersey, USA POSTER PRESENTATION

[24]. Nanoelectrospray high resolution mass spectrometry for the discovery of glycolipid biomarkers in neurodevelopmental disorders Petrut A., Sarbu M., Ica R., Munteanu C.V.A., Zamfir A. D., 16th International Conference on Nanosciences & Nanotechnologies (NN19), 1-5 july 2019, Salonic, Greece –ORAL PRESENTATION.

[25]. Development of chip-based mass spectrometry for structural and functional protein-brain ganglioside interactions Sarbu M., Popescu L., Ica R., Petrut A., Zamfir A.D.,16th International Conference on Nanosciences & Nanotechnologies (NN19), 1-5 july 2019, Salonic, Greece – POSTER PRESENTATION.

[26]. Determination of the structure and functions of glycophingolipids in human nucleus caudatus by Orbitrap multistage mass spectrometry, Sarbu M., Ica R., Petrut A., Munteanu C.V.A., Petrescu A.J., Zamfir A.D., 16th International Conference on Nanosciences & Nanotechnologies (NN19), 1-5 july 2019, Salonic, Greece – POSTER PRESENTATION.

[27]. Mapping and sequencing of ganglioside in cerebellum by nanoelectrospray high resolution tandem mass spectrometry, Ica R., Sarbu M., Petrut A., Munteanu C.V.A., Petrescu A.J., Zamfir A.D., 16th International Conference on Nanosciences & Nanotechnologies (NN19), 1-5 july 2019, Salonic, Greece – POSTER PRESENTATION.

[28]. Determination of protein-brain ganglioside interactions by chip-based nanoelectrospray quadrupole-time-of-flight tandem mass spectrometry, Popescu L., Sarbu M., Ica R., Petrut A., Zamfir A.D., 67th ASMS Conference on Mass Spectrometry and Allied Topics, 2-6 june 2019, Atlanta, SUA – POSTER PRESENTATION.

[29]. Ganglioside biomarker discovery and characterization in neurodevelopmental diseases by high resolution multistage mass spectrometry, Sarbu M., Ica R., Munteanu C.V.A., Petrut A., Zamfir A.D., 67th ASMS Conference on Mass Spectrometry and Allied Topics, 2-6 june 2019, Atlanta, SUA – POSTER PRESENTATION.

[30]. Mapping and sequencing of gangliosides in human cerebellum at different developmental stages by Orbitrap multistage mass spectrometry, Ica R., Sarbu M., Petrut A., Munteanu C.V.A., Petrescu A.J., Albulescu R., Zamfir A.D., 67th ASMS Conference on Mass Spectrometry and Allied Topics, 2-6 june 2019, Atlanta, SUA – POSTER PRESENTATION.

[31]. Determination of ganglioside composition, structure and functional interactions in human brain in health and disease by advanced mass spectrometry, Sarbu M., Ica R., Petrut A., Munteanu C., Clemmer D.E., Zamfir A.D., 8th International Symposium Research and Education in Innovation Era, 23-25 may 2019, Arad, Romania – ORAL PRESENTATION.

[32]. . Human gangliosidome in severe brain disorders: high resolution mass spectrometry study, Ica R., Sarbu M., Cozma I., Petrescu A.J., Munteanu C.V.A., Zamfir A.D., 17th Student scientific session, 11-13 April 2019, Arad, Romania – ORAL PRESENTATION .

[33]. Function and structural protein-glycolipid interaction by high performance mass spectrometry, Secara A., Ica R., Sarbu M., Popescu L., Petrut A., Petrica L., Zamfir A.D., 17th Student scientific session, 11-13 April 2019, Arad, Romania – POSTER PRESENTATION .

[34].Ganglioside expression and structure in normal human cerebellum: an Orbitrap mass spectrometry survey, Ica R., Sarbu M., Munteanu C.V.A., Petrut A., Petrescu A.J., Albulescu R., Zamfir A.D., 30th MassSpec Forum Vienna, 16-22 February 2019, Viena, Austria –POSTER PRESENTATION.

[35].High resolution mass spectrometry provides novel insights into the structure and functions of gangliosides expressed in nucleus caudatus, Sarbu M., Popescu L., Ica R., Munteanu C.V.A., Petrut A., Petrescu A.J., Zamfir A.D., 30th MassSpec Forum Vienna, 16-22 February 2019, Viena, Austria – POSTER PRESENTATION.

[36].An advanced mass spectrometry approach for glycolipid biomarker discovery in neurodevelopmental diseases, Sarbu M., Ica R., Munteanu C.V.A., Petrescu A.J., Petrut A., Zamfir A.D., 30th MassSpec Forum Vienna, 16-22 February 2019, Viena, Austria – POSTER PRESENTATION.

[37].Fully automated chip-based nanoESI analyses of protein-brain ganglioside interactions, Popescu L., Sarbu M., Ica R., Petrut A., Zamfir A.D., 30th MassSpec Forum Vienna 2019, 16-22 February 2019, Viena, Austria – POSTER PRESENTATION.

[38]. Ion mobility mass spectrometry and chip mass spectrometry analyses for functional and structural interactions of gangliosides, Zamfir A.D., Sarbu M., Ica R., Albulescu R., Petrescu A.J., Walsh Seminar, 16-23 January 2019, New York, USA – ORAL PRESENTATION.

[39]. Screening and sequencing of lipid-linked carbohydrates in human caudate nucleus by nanoelectrospray ionization high resolution tandem mass spectrometry. Alina petrut , Cristian V.A. Munteanu , Mirela Sarbu , Raluca Ica , Alina D. Zamfir , Andrei J. Petrescu. RSBMB International Conference 2019, Iasi Romania 26-27 September. POSTER PRESENTATION.

[40]. Development of advanced mass spectrometric platforms for glycoconjugate mapping, biomarker discovery and study of their functional interactions. Alina D. Zamfir , Mirela Sarbu , Alina Petrut, Raluca Ica, Cristian V.A. Munteanu, Anca Simulescu, Andrei J. Petrescu. RSBMB International Conference 2019 Iasi Romania 26-27 September. ORAL PRESENTATION

[41]. An advanced mass spectrometry approach for glycolipid biomarker discovery in neurodevelopmental diseases Mirela sarbu, Cristian V.A. Munteanu, Raluca Ica, Alina Petrut, Andrei J. Petrescu, Alina D. Zamfir. RSBMB International Conference 2019 Iasi Romania 26-27 September. POSTER PRESENTATION.

[42]. Glycosphingolipidomics of human cerebellum by orbitrap multistage mass spectrometry. Raluca Ica, Cristian V.A. Munteanu, Alina Petrut, Mirela Sarbu, Andrei J. Petrescu, Alina D. Zamfir. RSBMB International Conference 2019 Iasi Romania26-27 September. POSTER PRESENTATION.

[43]. Development of modern high resolution mass spectrometry platform for glycolipid mapping in defined human brain regions. Alina Petrut, Anca Simulescu, Raluca Ica, Mirela Sarbu, Cristian V.A. Popescu, Alina D. Zamfir, Andrei J. Petrescu. 25rd International Symposium on Analytical and Environmental Problems, 7-8 Octomber 2019, Szeged, Hungary – POSTER PRESENTATION .

[44]. Determination of structural-functional interactions of gangliosides with peptides and proteins by microfluidics –mass spectrometry Raluca Ica, Mirela Sarbu, Alina Secara, Laurentiu Popescu, Alina Petrut, Alina D. Zamfir. 25rd International Symposium on Analytical and Environmental Problems, 7-8 October 2019, Szeged, Hungary – POSTER PRESENTATION.

[45]. Advanced mass spectrometry techniques for determination of brain ganglioside expression and their functional interactions in healthy and diseased central nervous system. Mirela Sarbu, Raluca Ica, Alina Petrut, Cristian V.A. Munteanu, David E. Clemmer, Alina D. Zamfir. 25rd International Symposium on Analytical and Environmental Problems, 7-8 October 2019, Szeged, Hungary – ORAL PRESENTATION.