An innovative analytical platform integrating nanoelectrospray ionization (nanoESI), ion mobility mass spectrometry (IMS MS) and tandem MS fragmentation (MS/MS) was designed and optimized to address cerebrospinal fluid (CSF)-specific glycosphingolipidom for the discovery of biomarkers, as well as for monitoring changes in the expression of glycosphingolipids (GSL) that occur during the development of central nervous system (CNS) pathologies. The nanoESI IMS MS platform was successfully tested, implemented and validated in the laboratory for the investigation of GSL mixtures extracted from healthy and diseased human CSF . Last but not least, the nanoESI IMS MS and MS/MS platform was optimized for the separation, fragmentation and structural analysis of components in very complex native mixtures of CSF.
As a final step, a complete inventory was made with new glycosphingolipid species discovered in the CSF within the CSFIMS project and postulated to be associated (molecular marker) with a certain brain pathology, especially tumor markers.
A challenge for modern biomedical and biophysical methods is the continuous development and improvement of existing investigation methods able to decipher all the changes that occur at the molecular level. Thus, the proposed research aims the implementation in specialized CSF screening test as a method of early diagnosis in incipient stages of CNS pathologies development, as well as for monitoring treatment effectiveness, of IMS MS for the identification of the specific sialylated and sulfated GSL biomarkers settled upon comparative studies on healthy and diseased CSF. Within the multidisciplinary CSFIMS project, the implementation of the novel concepts pursue to open new research directions in the biomedical analysis, in particular in glycolipidomics field, by implementing the most powerful method in the research of CSF. This approach will be able to provide a high-throughput characterization in order to decipher the structure-function relationship for each GG and ST species involved in a physiological/pathological process, finally leading to brain cancer biomarker discovery and the establishment of a diagnosis in an early stage of disease.
Stage 1 (Ianuarie-December 2021)
Development of IMS MS and MS/MS for screening and sequencing of sialylated and sulfated GSLs (GGs and STs) derived from CSF of healthy individuals, of different ages.
Given the complexity and low concentration of GSL in the CSF, to obtain maximum information on the composition and structure of the analyzed species, at this stage in the first technical phase nanoESI QTOF MS characterized by high resolution will be developed and optimized for investigating GSL complexes from CSF of healthy people. After developing the protocol and setting the optimal parameters, the IMS MS and MS/MS technique will be developed to characterize GSL complexes in the CSF, a technique that ensures the separation of ions according to their mobility, fragmentation and detection of molecular ions and fragment ions in a single experiment.
Activities in Stage 1:
A1.1. Optimization of high resolution MS and CID MS/MS for screening and sequencing of GSLs derived from healthy CSF.
A1.2. IMS MS for separation and screening of GSLs derived from healthy CSF.
A1.3. Structural characterization by IMS CID MS/MS of GSLs derived from healthy CSF.
Stage 2 (January-December 2022)
Development of IMS MS and MS/MS for screening and sequencing of sialylated and sulfated GSLs derived from CSF of individuals with astrocytoma and glioblastoma brain cancers.
The GSL complexes originating from CSF of patients diagnosed with astrocytoma and other glioblastoma multiforme will be screened by IMS MS to provide valuable data upon the modification that occur during malignant transformation by comparing the CSF profile with the one determined by QTOF MS. The newly identified species with possible biomarker role will be structurally characterized by CID MS/MS for isomers and conformers discrimination and their structural elucidation and further, introduced in our human glycosphingolipidome database.
Activities in Stage 2:
A2.1. Optimization of high resolution MS and CID MS/MS for screening and sequencing of GSLs derived from diseased CSF.
A2.2. IMS MS for separation and screening of GSLs derived from diseased CSF
A2.3. Structural characterization by IMS CID MS/MS of GSLs derived from diseased CSF.
1. Ion mobility mass spectrometry reveals the complexity of ganglioside structures expressed in human cerebrospinal fluid, Sarbu M, Clemmer DE, Zamfir AD, 2nd Advanced Chemistry World Congress, Berlin (Germania) 14th-15th June 2021, POSTER;
2. Ion mobility mass spectrometry reveals novel and rare ganglioside structures in human cerebrospinal fluid, Sarbu M, Vukelić Ž, Clemmer DE, Zamfir AD, 45th FEBS Congress, Ljubljana (Slovenia) 3rd-8th July 2021, POSTER;
3. Brain glycomics by novel mass spectrometry approaches based on ion mobility and chip-nanoelectrospray ionization, Zamfir AD, Sarbu M, Ica R, Clemmer DE, Vukelić Ž, 45th FEBS Congress, Ljubljana (Slovenia) 3rd-8th July 2021, INVITED LECTURE;
4. GalNAc-GD1 is a biomarker of cerebrospinal fluid as revealed by nanoESI ion mobility mass spectrometry, Sarbu M, Clemmer DE, Zamfir AD, 18th International Conference on Nanosciences and Nanotechnologies, Thessaloniki (Greece) 6th-9th July 2021, POSTER;
5. Ion mobility mass spectrometry of human glioblastoma gangliosides, Sarbu M, Petrica L, Clemmer DE, Vukelić Ž, Zamfir AD, 69th ASMS Conference on Mass Spectrometry and Allied Topics, Philadelphia (USA) 31st October-4th November 2021, POSTER;
6. Ion mobility separation mass spectrometry reveals the occurrence in human cerebrospinal fluid of atypical GalNAc-GD1c glycoforms, Sarbu M, Clemmer DE, Zamfir AD, 69th ASMS Conference on Mass Spectrometry and Allied Topics, Philadelphia (USA) 31st October-4th November 2021, POSTER;
7. Glycosphingolipidomics of human glioblastoma multiforme by ion mobility tandem mass spectrometry, Sarbu M, Clemmer DE, Vukelić Ž, Zamfir AD, 70th ASMS Conference on Mass Spectrometry and Allied Topics, Minneapolis (USA) 4th-9th June 2022, POSTER;
8. IMS MS of human cerebrospinal fluid: novel insights into the structure of ganglioside isomers identified de novo, Sarbu M, Fabris D, Vukelić Ž, Clemmer DE, Zamfir AD, 32nd Mass Spectrometry Forum, Viena (Austria) 5th-6th July 2022, POSTER;
9. Nanoelectrospray ionization ion mobility mass spectrometry for biomarker discovery in malignant brain tumors, Sarbu M, Vukelić Ž, Clemmer DE, Zamfir AD, 19th International Conference on Nanosciences and Nanotechnologies, Thessaloniki (Greece) 5th-8th July 2022, POSTER;
10. Development and application of ion mobility tandem mass spectrometry for the investigation of human cerebrospinal fluid gangliosidome, Sarbu M, Fabris D, Vukelić Ž, Clemmer DE, Zamfir AD, 24th International Mass Spectrometry Conference, Maastricht (Netherlands) 27th August – 2nd September 2022, ORAL PRESENTATION;
11. Screening and sequencing of sialylated glycosphingolipids in human glioblastoma by ion mobility mass spectrometry, Sarbu M, Clemmer DE, Vukelić Ž, Zamfir AD, 28th International Symposium on Analytical and Environmental Problems, Szeged (Hungary) 14th-15th November 2022, POSTER.
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 (IF 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 (IF 3,02)
3. Gangliosides as biomarkers of human brain diseases: trends in discovery and characterization by high-performance mass spectrometry, Sarbu M, Ica R, Zamfir AD, Int J Mol Sci 2022, 23, 693 (IF 6.208)
4. Ion mobility mass spectrometry reveals rare sialylated glycosphingolipid structures in human cerebrospinal fluid Sarbu M, Fabris D, Vukelić Ž, Clemmer DE, Zamfir AD, Molecules 2022, 27, 743 (IF 4.927)
5.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 2022 (accepted) (IF 3.595)
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.
Project Number: PD 38/2020
Project Director: CS Dr. Mirela Sarbu
Project Title:Development of ion mobility mass spectrometry for screening and sequencing of sialylated and sulfated glicosphingolipids in cerebrospinal fluid in health and disease Acronym: CSFIMS