CHON-DERM-IMS proposal aims to develop a robust ion mobility mass spectrometry (IMS MS) and tandem MS (MS/MS) by collision-induced dissociation (CID) methodology for the analysis of irregularly sulfated chondroitin/dermatan sulfate (CS/DS) domains and their interactions, which would lead to: i) discovery of new biologically active CS/DS sequences; ii) the first introduction of IMS MS in glycomics of hybrid CS/DS domains; iii) the first IMS MS application to brain glycosaminoglycomics.
The project is structured in 4 work packages:
1. Development of IMS MS and CID MS/MS for compositional and structural analysis of CS/DS oligosaccharides. Discovery and detailed characterization of irregularly sulfated CS/DS domains in human decorin and biglycan;
2. Human brain CS/DS: identification and structure elucidation of regularly and irregularly sulfated motifs by IMS MS and CID MS/MS;
3. Determination of CS/DS-protein interactions by IMS MS and CID MS/MS;
4. Dissemination of the results.
It is expected that the novel approach proposed here will enable the detection and structural investigation of under- and oversulfated CS/DS motifs in various proteoglycans, including those expressed in the central nervous system (much less investigated so far) and the identification of active sequences on the basis of their interactions with growth factors, IMS separation followed by MS profiling and detailed structural analysis by MS/MS fragmentation of the formed noncovalent complexes.
IMS MS and CID MS/MS methodology optimized for separation, detection and structural analysis will be applied to hybrid CS/DS fractions from human skin fibroblast decorin (DCN), biglycan (BGN) and human brain. The estimated results are: i) validation of the developed IMS MS protocols for CS/DS mixtures; ii) discovery and characterization of misregulations in the sulfation of CS/DS motifs; iii) the first introduction of IMS MS in brain glycosaminoglycomics; iv) discovery of novel biologically active CS/DS domains in DCN, BGN and human brain; v) structural elucidation of the reaction products following CS/DS interactions with protein growth factor-2 (FGF-2).
The general objective of CHON-DERM-IMS project is to conceive, develop to the stage of routine use and introduce a novel and robust methodology in the field of functional glycomics of hybrid CS/DS oligosaccharides. The methodology to be developed for this purpose is based on high-throughput IMS MS in combination with the highly efficient fragmentation technique: collision-induced dissociation (CID) for detailed structural analysis of biologically active sequences, determination of the CS/DS noncovalent complexes with proteins and their detailed structural characterization by top-down fragmentation.
Specific objectives
a) Development of a novel approach based on IMS MS for separation of CS/DS structural isomers, detection of CS/DS in mixtures of short and long chains of different sulfation pattern and discrimination of isobaric structures; b) Development of novel protocols for high throughput IMS CID MS/MS fragmentation of under- and oversulfated hybrid CS/DS oligosaccharides in data-dependent analysis (DDA) mode; c) Designing of IMS MS-based interaction assays, to allow for the first time a detailed assessment of the formed noncovalent complexes, with focus on the exact localization of the sulfate groups, determined by top-down fragmentation of the intact CS/DS-protein aggregate. The IMS CID MS/MS protocols will be for the first time developed, tested and implemented in functional glycosaminoglycomics to provide fast and accurately the key structural elements of the complexes at high sensitivity and in a single run: IMS separation of the formed complexes, MS detection, MS/MS fragmentation.
IMS MS and CID MS/MS methodology optimized for separation, detection and structural analysis will be applied to hybrid CS/DS fractions from human skin fibroblast decorin (DCN), biglycan (BGN) and human brain. The estimated results are: i) validation of the developed IMS MS protocols for CS/DS mixtures; ii) discovery and characterization of misregulations in the sulfation of CS/DS motifs; iii) the first introduction of IMS MS in brain glycosaminoglycomics; iv) discovery of novel biologically active CS/DS domains in DCN, BGN and human brain; v) structural elucidation of the reaction products following CS/DS interactions with protein growth factor-2 (FGF-2).
1. Developments and applications of chip-based mass spectrometry in glycomics of chondroitin/dermatan sulfate oligosaccharides, A.D. Zamfir, 2nd Advanced Chemistry World Congress, Berlin (Germania) 14-15 june 2021, INVITED LECTURE;
2. Glycolipidomics of human brain hemangioma by high resolution multistage mass spectrometry, Raluca Ica, Mirela Sarbu, Cristian V.A. Munteanu, Alina D. Zamfir, 2nd Advanced Chemistry World Congress, Berlin (Germania) 14-15 june 2021, POSTER;
3. Brain glycomics by novel mass spectrometry approaches based on ion mobility and chip-nanoelectrospray ionization, Alina D. Zamfir, Mirela Sarbu, Raluca Ica, David E. Clemmer, Zeljka Vukelić, 45th FEBS Congress, Ljubljana (Slovenia) 3-8 july 2021, INVITED LECTURE;
4. Discovery of novel biomarkers in human brain hemangioma by advanced mass spectrometric methods, R. Ica, M. Sarbu, Ž. Vukelić, AD. Zamfir, 45th FEBS Congress, Ljubljana (Slovenia) 3-8 july 2021, POSTER;
5. GalNAc-GD1 is a biomarker of cerebrospinal fluid as revealed by nanoESI ion mobility mass spectrometry, M. Sarbu, D.E. Clemmer, A.D. Zamfir, 18th International Conference on Nanosciences and Nanotechnologies (NN21), Salonic (Grecia), 6-9 july 2021, POSTER;
6. Nanoelectrospray ionization high resolution mass spectometry of glycolipids expressed in human cortex, R. Ica, Ž. Vukelić, A. D. Zamfir, 18th International Conference on Nanosciences and Nanotechnologies (NN21), Salonic (Grecia), 6-9 july 2021, POSTER;
7. Determination of human motor cortex gangliosidome by nanoelectrospray high resolution multistage mass spectrometry, R. Ica, C. V.A. Munteanu, Ž. Vukelić, A.D. Zamfir, 69th ASMS Conference on Mass Spectrometry and Allied Topics, Philadelphia (USA), 31 October- 4 November 2021, POSTER;
8. Ion mobility mass spectrometry of human glioblastoma gangliosides, M. Sarbu, L. Petrica, D.E. Clemmer, Ž. Vukelić, A.D. Zamfir, 69th ASMS Conference on Mass Spectrometry and Allied Topics, Philadelphia (USA), 31 October-4 Novembre 2021, POSTER;
9. Ion mobility separation mass spectrometry reveals the occurrence in human cerebrospinal fluid of atypical GalNAc-GD1c glycoforms, M. Sarbu, D.E. Clemmer, A.D. Zamfir, 69th ASMS Conference on Mass Spectrometry and Allied Topics, Philadelphia (USA), 31 October-4 November 2021, POSTER;
10. Development of microfluidics-mass spectrometry for structural analysis of chondroitin/dermatan sulfate oligosaccharides, A.D. Zamfir, M. Sarbu, R. Ica, D. Seidler, 18th International Conference on Nanosciences & Nanotechnologies (NN21), Salonic (Grecia), 6-9 july 2021, POSTER;
1. Discovery and characterization of irregularly sulfated chondroitin/dermatan sulfate domains in human decorin by ion mobility tandem mass spectrometry, Raluca Ica, Edie M. Sharon, M. Sarbu, David E. Clemmer, Alina D. Zamfir, 70th ASMS Conference on Mass Spectrometry and Allied Topics (ASMS 2022), 5-9 iunie 2022 (Minneapolis, MN, USA) POSTER
2. Determination of gangliosides associated to temporal lobe epilepsy by high resolution tandem mass spectrometry, R. Ica, K. Mlinac-Jerković, K. Ilić, T. Sajko, AD. Zamfir, S. Kalanj-Bognar, 70th ASMS Conference on Mass Spectrometry and Allied Topics (ASMS 2022), 5-9 iunie 2022 (Minneapolis, MN, USA) POSTER
3. IMS MS reveals novel hybrid chodroitin/dermatan sulfate domains in human decorin, Mirela Sarbu, Raluca Ica, Roxana Biricioiu, David E. Clemmer, Alina D. Zamfir, 32 nd Mass Spectrometry Forum 2022 (Viena, Austria, 5-6 iulie 2022), POSTER
4. Characterization by high resolution mass spectometry of glycolipids expressed in human cortex, R. Ica, Ž. Vukelić, A. D. Zamfir, 32nd Mass Spectrometry Forum 2022 (Viena, Austria, 5-6 iulie 2022) POSTER
5. Nanoelectrospray ionization ion mobility mass spectrometry for biomarker discovery in malignant brain tumors, M. Sarbu, Ž. Vukelić, D.E Clemmer, A.D. Zamfir, 19th International Conference on Nanosciences & Nanotechnologies (NN22), 5-8 iulie 2022, Salonic, Grecia POSTER
6. Nanoelectrospray ion mobility mass spectrometry for the analysis of chondroitin/dermatan sulfate oligosaccharides, R. Ica, M. Sarbu, E. M. Sharon, David E. Clemmer, Alina D. Zamfir, 19th International Conference on Nanosciences &; Nanotechnologies (NN22), 5-8 iulie 2022, Salonic, Grecia, POSTER
7. Implementation of advanced mass spectrometry methods based on nanoelectrospray for glycolipidome analysis in neurological diseases, R. Ica, C.V.A. Munteanu, S. Kalanj-Bognar, A.D. Zamfir, 19th International Conference on Nanosciences &; Nanotechnologies (NN22), 5-8 iulie 2022, Salonic, Grecia, POSTER
8. Combining size-exclusion chromatography and ion mobility mass spectrometry for the identification of chondroitin/dermatan sulfate octamers in decorin, Mirela Sarbu, Raluca Ica, Edie M. Sharon, David E. Clemmer, Alina D. Zamfir, 24th International Mass Spectrometry Conference (IMSC 2022), 27 august - 2 septembrie (Maastricht, Olanda), POSTER
9. Characterization of hybrid chondroitin/dermatan sulfate octasaccharide domains in human brain by ion mobility tandem mass spectrometry, M. Sarbu, R. Ica, R. Biricioiu, A. Crumpei, E. Sharon, David E. Clemmer, Alina D. Zamfir, 28th International Symposium on Analytical and Environmental Problems, 14-15 noiembrie 2022, Szeged, Ungaria
1. Developments and applications of separation and microfluidics methods coupled to electrospray mass spectrometry in glycomics of nervous system gangliosides, Sarbu M, Ica R, Zamfir AD, Electrophoresis 2021, 42, 429-449. Factor de impact 3.53
2. Gangliosides of human glioblastoma multiforme: a comprehensive mapping and structural analysis by ion mobility tandem mass spectrometry, • Sarbu M, Petrica L, Clemmer DE, Vukelić Ž, Zamfir AD, J Am Soc Mass Spectrom.2021, 32, 1249-1257. Factor de impact 3.02.
3. High resolution mass spectrometry reveals a complex ganglioside pattern and novel polysialylated structures associated to human motor cortex, Ica R, Munteanu CVA, Vukelić Ž, Zamfir AD, Eur J Mass Spectrom (Chichester)2021 Sep 13; 14690667211040912. DOI: 10.1177/14690667211040912. Factor de impact 1.06.
Book Chapter
1. Modern Techniques for Separation, Mass Spectrometric Detection and Characterization of Glycolipids, Sarbu M, Zamfir AD, in Carbohydrate Analysis by Modern Liquid Phase Separation Techniques 2nd Edition (Editor, Ziad El Rassi), Elsevier, eBook ISBN: 978-012-824-5125; Paperback ISBN: 978-012-821-4473, pp. 485-527, 2021
1. Identification and Structural Characterization of Novel Chondroitin/Dermatan Sulfate Hexassacharide Domains in Human Decorin by Ion Mobility Tandem Mass Spectrometry Sarbu M, Ica R, Sharon E, Clemmer DE, Zamfir AD. Molecules 2022, 27, 6026. Factor de impact 4.927
2. Gangliosides as biomarkers of human brain diseases: trends in discovery and characterization by high-performance mass spectrometry Sarbu M, Ica R, Zamfir AD. J. Mol. Sci. 2022, 23(2):693. doi: 10.3390/ijms23020693. Factor de impact 6.208
3. Capillary zone electrophoresis-electrospray ionization tandem mass spectrometry for total analysis of chondroitin/dermatan sulfate oligosaccharides Zamfir AD Methods Mol. Biol. 2022;2531:163-184. Factor de impact 1.37.
4. Ganglioside analysis in body fluids by liquid-phase separation techniques hyphenated to mass spectrometry Suteanu-Simulescu A, Sarbu M, Ica R, Petrica L, Zamfir AD Electrophoresis (accepted) 2022, Factor de impact 3.595.
5. Ion mobility tandem mass spectrometry characterization of disaccharide domain in human biglycan Sarbu M, Ica R, Sharon E, Clemmer D, Zamfir AD Proteomes (submitted) 2022
6. High-Resolution Tandem Mass Spectrometry Identifies a Particular Ganglioside Pattern in Early Diabetic Kidney Disease of Type 2 Diabetes Mellitus Patients Suteanu-Simulescu A, Zamfir AD, Ica R, Sarbu M, Munteanu CVA, Gadalean F, Vlad A, Bob F, Jianu DC, Petrica L Molecules 2022 doi: 10.3390/molecules27092679 Factor de impact 4.927
7. Ion Mobility Mass Spectrometry Reveals Rare Sialylated Glycosphingolipid Structures in Human Cerebrospinal Fluid Sarbu M, Fabris D, Vukelic Ž, Clemmer DE, Zamfir AD Molecules 2022, doi: 10.3390/molecules27030743 Factor de impact 4.927
Stage 1 (January 2021-December 2021)
Development of IMS MS and CID MS/MS for compositional and structural analysis of CS/DS oligosaccharides. Discovery and detailed characterization of irregularly sulfated CS/DS domains in human decorin and biglycan.
The first tasks are related to the development of IMS and CID MS/MS for separation, detection and fragmentation of CS/DS oligosaccharides and the allied optimization procedures. The optimization in the negative ion mode will be carried using commercially available fractions (dp2, dp4) and will be focused on the IMS separation according to the sulfation degree and discrimination of isobaric structures. The optimized methodology will be applied for: i) IMS separation and MS screening of hybrid di- to decasaccharides from human DCN and BGN; ii) detailed structure elucidation by MS/MS of irregularly sulfated species, i.e. the ions, which, according to exact mass determination correspond to structures bearing more (or less) than one sulfate group per disaccharide unit, since such species (in particular the oversulfated ones) are of major interest as possible active sequences.
Activities within Stage 1
A1.1 Optimization of IMS separation and nanoESI MS detection of CS/DS by using standard CS and DS fractions of known sulfation pattern.
A1.2 Optimization of IMS separation followed by CID MS/MS fragmentation of CS and DS oligosaccharides by using standard fractions of known sulfation pattern.
A1.3 IMS and CID MS/MS compositional and detailed structural analysis of hybrid CS/DS fractions (dp2 to dp10) from conditioned media of cultured human skin fibroblasts DCN.
A1.4. IMS and CID MS/MS compositional and detailed structural analysis of hybrid CS/DS fractions (from dp2 to dp10) from HEK293 cell line BGN.
Stage 2 (January 2022-December 2022)
Human brain CS/DS: identification and structure elucidation of regularly and irregularly sulfated motifs by IMS MS and CID MS/MS.
In the second stage of the project the methodology developed in the first stage will be applied to CS/DS released from human brain. For testing the method performance and sensitivity for brain CS/DS, individual dp2 to dp10 brain CS/DS fractions will be subjected to IMS MS separation, mapping, identification of unusually sulfated species according to their exact m/z measurement followed by CID MS/MS of domains exhibiting misregulation in the sulfation pattern. In the second stage, brain CS/DS mixtures resulting after chain digestion with Chon B lyase and AC I lyase will be separated by IMS MS. Potential binding domains i.e. species of misregulated sulfation will be characterized by CID MS/MS in terms of epimerization and sulfation code: number of sulfate groups and determination of the sulfation sites.
Activities within Stage 2
A2.1 IMS MS of individual hybrid CS/DS fractions (from disaccharide- dp2 to decasaccharide-dp10) extracted from human brain.
A2.2. IMS CID MS/MS detailed structural analysis of individual hybrid CS/DS domains (from dp2 to dp10) extracted from human brain.
A2.3. IMS MS profiling and fragmentation analysis by CID MS/MS of brain CS/DS oligosaccharide mixture resulted after depolymerization with Chon B.
A2.4. IMS MS profiling and fragmentation analysis by CID MS/MS of complex brain CS/DS oligosaccharide mixture resulted after depolymerization with Chon AC.
Stage 3 (January 2023-December 2023)
Determination of CS/DS-protein interactions by IMS MS and CID MS/MS.
The aim of this research stage is the study of the noncovalent interactions CS/DS-FGF-2 by IMS CID MS/MS and structural characterization of the active sequences. Several buffer systems mimicking the in vivo environment will be tested in terms of compatibility with the interaction and IMS MS analysis. FGF-2, DCN-, BGN- and brain-derived CS/DS obtained after digestion with Chon B and independently with Chon AC I lyase will be dissolved in the buffer and analyzed each by IMS MS and CID MS/MS. To follow the interaction dynamics, the in vitro assays will include incubation of the CS/DS and the FGF-2 followed by IMS MS of aliquots collected at different time intervals. The detected CS/DS-FGF-2 complexes will be structurally characterized in CID MS/MS experiments, with a special focus on the complexes formed with long CS/DS chains.
Activities within Stage 3
A3.1 IMS MS and CID MS/MS screening and fragmentation analysis of FGF-2 in different aq. buffer systems.
A3.2 IMS MS and CID MS/MS screening and fragmentation analysis of CS/DS oligosaccharides in different aq. buffer systems.
A3.3 Binding assay: IMS MS and CID MS/MS determination and characterization of FGF-2 protein interactions with CS/DS from DCN and BGN.
A3.4 Binding assay: IMS MS and CID MS/MS of FGF-2 protein interactions with CS/DS oligosaccharides from human brain.
Project Number: PN-III-P4-ID-PCE-2020-0209
Project Director: CS 1 Prof. Dr. Fiz. Alina-Diana Zamfir
Project Title:Discovery and characterization of irregularly sulfated chondroitin/dermatan sulfate domains and their interactions by ion mobility mass spectrometry (Acronym: CHON-DERM-IMS)