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.

Conference presentations

[1]. Profiling and structural characterization by mass spectrometry of region-specific gangliosides in brain M. Sarbu, Z?. Vukelicī, D.E. Clemmer, A.D. Zamfir 23rd International Symposium on Analytical and Environmental Problems, 9-10 October 2017, Szeged, Ungaria 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, Ungaria 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, Ž. 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, Z?eljka Vukelicī, 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, Z?eljka Vukelicī, 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, Z?eljka Vukelicī, 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, Z?eljka Vukelicī, 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, Z?eljka Vukelicī 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, Z?eljka Vukelicī, 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, Z?eljka Vukelicī, David E. Clemmer, Alina Diana Zamfir 12th Central and Eastern European Proteomic Conference (CEEPC), 12-14 November 2018, New Jersey, USA POSTER PRESENTATION