In MICROIMS project a novel bioanalytical platform, integrating microfluidics for electrospray ionization (ESI) and ion mobility mass spectrometry (IMS MS), will be designed, introduced in human brain glycolipidomics and validated for biomarker discovery.

The system to be developed is based on the coupling of fully automated chip-nanoESI with IMS MS and tandem MS (MS/MS). In the first stage of research IMS MS and MS/MS will be developed in the negative ion mode and optimized for simple mixtures. The system will be validated on ganglioside fractions isolated from human brain. The second stage of research will be focused on the platform development for highly complex mixtures. Native ganglioside mixtures extracted from human adult and fetal brain biopsies will be analyzed in IMS and CID MS/MS modes for compositional and structural investigation and biomaker discovery. Following the data mining and interpretation, we will complete with de novo identified species our database of structures forming the human brain gangliosidome. The purpose of the last research stage is platform validation for glycolipid biomarker discovery in brain tumors and neurdegeneration using native ganglioside extracts from primary benign and malignant brain tumors and neurodegenerative disease, the final goal being the in-laboratory platform validation for disease-related glycolipid biomarker discovery. The last step of the research will be dedicated to completing brain tumor gangliosidome database with the newly discovered structures and proposals of biomarkers.

The general objective of MICROIMS project is to design, introduce in the field of human brain glycolipidomics and in-laboratory validate for biomarker discovery of a novel and robust analytical platform integrating microfluidics for electrospray ionization and ion mobility mass spectrometry. For this purpose a high-throughput fully automated chip-nanoESI system on a NanoMate robot will be on-line coupled to IMS MS and MS/MS, the latter being performed using the highly efficient fragmentation technique: CID. The entire on-line platform will be optimized for mapping and structural analysis of gangliosides in human brain and biomarker discovery.

Due to the combined high sensitivity, ionization and separation efficiency, the proposed platform, optimized for the investigation of brain sialylated glycolipids, has a high potential to provide better insights into the complexity of structures expressed in human brain than ever reported, and, most importantly to discover and structurally characterize new, previously not even detected, ganglioside species associated to severe brain pathologies.

The most efficient ionization system existing nowadays and the most efficient mass spectrometry system incorporating ion mobility separation at high resolution was integrated, powerful platform optimized and validated for screening and identification of complex mixtures of glycans/glycoconjugates extracted from human matrices and discovery of novel molecular markers.

(a) IMS separation of fetal brain gangliosides ionized by chip ;

(b) detection and determination of their mass by MS

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. Impact Factor 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. Impact Factor 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. Impact Factor 1.06.

4. High resolution mass spectrometry with chip-based ionization for the assessment of noncovalent interactions of proteins with normal brain and brain tumor gangliosides, Ica R, Sarbu M, Zamfir AD, Scien. Tech. Bull-Chem. Food Sci. Eng.Vol. 17 (XVIII), 2020, 15-19.

Book Chapter

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

2022

1. Gangliosides as Biomarkers of Human Brain Diseases: Trends in Discovery and Characterization by High-Performance Mass Spectrometry M Sarbu, R Ica , A D Zamfir INT J MOL SCIENCES 2022, 23(2), 1-46, Impact Factor 4,41.

2. Ion Mobility Mass Spectrometry Reveals Rare Sialylated Glycosphingolipid Structures in Human Cerebrospinal Fluid M Sarbu, D Fabris, Ž Vukelić, D Clemmer, A D Zamfir MOLECULES 2022, 27, 1-13, Impact Factor 4,93.

3. High-Resolution Tandem Mass Spectrometry Identifies a Particular Ganglioside Pattern in Early Diabetic Kidney Disease of Type 2 Diabetes Mellitus Patients A Suteanu-Simulescu, A D Zamfir, R Ica, M Sarbu, Munteanu CVA, F Gadalean, A Vlad, F Bob, D C Jianu, L Petrica MOLECULES 2022, 27, 1-17, Impact Factor 4,93.

4. Gangliosidome of a Human Hippocampus in Temporal Lobe Epilepsy Resolved by High-Resolution Tandem Mass Spectrometry R Ica, K Mlinac-Jerkovic, K Ilic, T Sajko, C V A Munteanu, A D Zamfir, S Kalanj-Bognar MOLECULES 2022, 27, 1-17, Impact Factor 4,93.

1. 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;

2. 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;

3. Discovery of novel biomarkers in human brain hemangioma by advanced mass spectrometric methods, Raluca Ica, Mirela Sarbu, Željka Vukelić, Alina D. Zamfir, 45th FEBS Congress, Ljubljana (Slovenia) 3-8 july 2021, POSTER;

4. 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), 3-10 july 2021, POSTER;

5. Determination of human motor cortex gangliosidome by nanoelectrospray high resolution multistage mass spectrometry, Raluca Ica, Cristian V.A. Munteanu, Željka Vukelić, Alina D. Zamfir, 69th ASMS Conference on Mass Spectrometry and Allied Topics, Philadelphia (USA), 31 October- 4 November 2021, POSTER;

6. 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;

7. 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;

2022

8. Glycosphingolipidomics of human glioblastoma multiforme by ion mobility tandem mass spectrometry, M. Sarbu, D.E. Clemmer, Ž. Vukelić, A.D. Zamfir, 70th ASMS Conference on Mass Spectrometry and Allied Topics, Minneapolis, SUA, 4-9 june 2022, POSTER;

9. Determination of gangliosides associated to temporal lobe epilepsy by high resolution tandem mass spectrometry, R. Ica, K. Mlinac-Jerković, K. Ilić, T. Sajko, A.D. Zamfir, S. Kalanj-Bognar, 70th ASMS Conference on Mass Spectrometry and Allied Topics, Minneapolis, SUA, 4-9 june 2022, POSTER;

10. IMS MS of human cerebrospinal fluid: novel insights into the structure of ganglioside isomers identified de novo, M. Sarbu, D. Fabris, Ž. Vukelić, D.E. Clemmer, A.D. Zamfir, 32nd Mass Spectrometry Forum 2022, 5-6 july 2022, POSTER;

11. Characterization by high resolution mass spectometry of glycolipids expressed in human cortex, R. Ica, Ž. Vukelić, A.D. Zamfir, 32nd Mass Spectrometry Forum 2022 5-6 july 2022, POSTER;

12. 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 july, POSTER;

13. 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 july, POSTER;

14. High resolution mass spectrometry analysis of human brain hemangioma gangliosidome, R. Ica, M. Sarbu, C.V.A. Munteanu, A.D. Zamfir, 24th International Mass Spectrometry Conference (IMSC 2022), 27 august-2 september 2022, POSTER;

15. Development and application of ion mobility tandem mass spectrometry for the investigation of human cerebrospinal fluid gangliosidome, M. Sarbu, D. Fabris, Ž. Vukelić, D.E. Clemmer, 24th International Mass Spectrometry Conference (IMSC 2022), 27 august-2 september 2022, ORAL PRESENTATION;

Stage 1 (November-December 2020)

Interfacing chip-nanoESI with QTOF IMS MS and CID MS/MS.

Development of the system in negative ion mode and optimization for simple mixtures. Testing the performance of the system for the analysis of isolated ganglioside fractions in the human brain.

At this stage chip-nanoESI will be coupled online with IMS MS, and the assembly will be developed and optimized for chip ionization, separation of ions according to their mobility, followed by fragmentation and detection of molecular ions and fragment ions in a single experiment. For the development of the system, simple fractions of gangliosides isolated and purified from the human brain will be used.

Activities in Stage 1:

A1.1. NanoESI chip-coupling with QTOF IMS MS. Development of the system and methodology in the negative ion mode for ionization, separation by IMS and detection of molecular ions, fragmentation and detection of product ions in simple mixtures.

A1.2. Optimization of chip-nanoESI IMS MS and CID MS / MS for the separation and detection of isolated ganglioside fractions in the human brain.

Stage 2 (January-December 2021)

Development of chip-nanoESI QTOF IMS MS and CID MS /MS in the negative ion mode for complex mixtures. Testing the performance of the system for the analysis of native gangliosides extracted from biopsies of the normal human brain.

In Stage 2 the newly developed analytical platform will be tested on native mixtures of gangliosides extracted from normal adult and fetal brain tissue. Following the interpretation of the data, we will complete with the newly identified species our database containing the structures of the normal brain gangliosidome.

Activities in Stage 2:

A2.1. Development of the fully automated chip-nanoESI IMS MS system for the separation, detection and identification of components in complex mixtures. Applications to native ganglioside extracts from the entire normal adult human brain and from different regions of the brain.

A2.2. Application and optimization of chip-nanoESI QTOF IMS MS for the mapping of gangliosides in the fetal brain and in different regions of the fetal brain.

A2.3. Chip-nanoESI IMS CID MS/MS optimization for separation, fragmentation and structural analysis of components in highly complex mixtures. Applications to gangliosides in the human adult and fetal brain.

A2.4. Data mining, processing and interpretation. Completing the database of normal human brain gangliosidome with the newly identified structures.

Stage 3 (January-November 2022)

Validation of the chip-nanoESI QTOF IMS MS and CID MS/MS method for the discovery of glycolipid biomarkers in brain tumors and neurodegenerative diseases.

In Stage 3 the system will be tested on ganglioside extracts from benign and malignant primary tumors of the brain and neurodegeneration, the final objective being the in- laboratory validation of the system for the discovery of glycolipid biomarkers. The last step of the research will be dedicated to completing the database related to meningioma, hemangioma and glioblastoma tumors with the discovered structures and biomarker proposals.

Activities in Stage 3:

A3.1. Chip-nanoESI IMS MS and CID MS/MS testing for ganglioside identification in benign tumors and structural analysis.

A3.2. Optimization and validation of the system and chip-nanoESI IMS MS and CID MS/MS methodology for the discovery of biomarkers of malignant transformation and neurodegeneration.

A3.3. Data mining, processing and interpretation. Completing the database of brain cancer gangliosidome and neurodegenerative diseases with the new biomarker structures identified.