Orbitrap mass spectrometry for monitoring the ganglioside pattern in human cerebellum development and aging

Abstract

We have developed here a superior approach based on high-resolution (HR) mass spectrometry (MS) for monitoring the changes occurring with development and aging in the composition and structure of cerebellar gangliosidome. The experiments were focused on the comparative screening and structural analysis of gangliosides expressed in fetal and aged cerebellum by Orbitrap MS with nanoelectrospray ionization (nanoESI) in the negative ion mode. The employed ultrahigh-resolution MS platform allowed the discrimination, without the need of previous separation, of 159 ions corresponding to 120 distinct species in the native ganglioside mixtures from fetal and aged cerebellar biopsies, many more than detected before, when MS platforms of lower resolution were employed. A number of gangliosides, in particular polysialylated belonging to GT, GQ, GP, and GS classes, modified by O-fucosylation, O-acetylation, or CH 3 COO − were discovered here, for the first time in human cerebellum. These components, found differently expressed in fetal and aged tissues, indicated that the ganglioside profile in cerebellum is development stage-and agespecific. Following the fragmentation analysis by high-energy collision-induced dissociation (HCD) tandem MS (MS/MS), we have also observed that the intimate structure of certain compounds has not changed during the development and aging of the brain, an aspect which could open new directions in the investigation of ganglioside biomarkers in cerebellar tissue.

Ion Mobility Mass Spectrometry Reveals Rare Sialylated Glycosphingolipid Structures in Human Cerebrospinal Fluid

“…As constituents of cell membrane microdomains, GGs contribute to extracellular biologic events, such as the modulation of membrane proteins and ion channels, cell–cell recognition, adhesion, cellular differentiation, growth and intra- and intercellular signaling [ 3 , 4 , 5 ]. Loss-of-function mutations in GG biosynthetic enzymes cause severe neurodegenerative disorders [ 3 , 6 , 7 , 8 , 9 ]; alterations in the composition and concentration of particular GGs might also occur with aging [ 10 , 11 , 12 ] and in common neurodegenerative conditions, such as Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, multiple sclerosis and epilepsy [ 13 , 14 ]. Therefore, GGs are not only markers for cells at certain developmental stages, but also prone to become targets of disease-associated antibodies [ 1 ].…”

An integrated Qual/Quan strategy for ganglioside lipidomics using high‐resolution mass spectrometry and Skyline software

“…However, nano‐flow HPLC is still not routinely used because of the complexity of instrumentation, low sample throughput, and poor reproducibility in quantitative assays 22 . Most of the nano flow applications involving GS have used direct infusion mode combined with off‐line sample preparation and purification steps 23 . Because of the ease of operation of HPLC systems and the availability of wide range of C18 columns, LMF rates at 3–50 μL/min are now routinely used in quantitative bioanalysis involving complex matrices.…”

“…22 Most of the nano flow applications involving GS have used direct infusion mode combined with off-line sample preparation and purification steps. 23 Because of the ease of operation of HPLC systems and the availability of wide range of C18 columns, LMF rates at 3-50 μL/min are now routinely used in quantitative bioanalysis involving complex matrices. However, in our view, high-throughput ganglioside quantification, particularly under ESI(−) mode at LMF (1-30 μL/min),…”

“…steps 23. Because of the ease of operation of HPLC systems and the availability of wide range of C18 columns, LMF rates at 3-50 μL/min are now routinely used in quantitative bioanalysis involving complex matrices.…”

High-resolution mass spectrometry reveals a complex ganglioside pattern and novel polysialylated structures associated with the human motor cortex

“…Although gangliosides are found in several organs and body fluids, such as blood, cerebrospinal fluid, saliva and urine, the most abundantly expressed species were discovered in the central nervous system 13 − 15 . In human brain, the expression and structure of gangliosides exhibits a well-defined regional specificity which changes, also in a specific way, with age 16 and when the respective brain area is affected by neuropathologies including genetic disorders, neurodegenerative diseases, benign or malignant tumors. 17 − 20…”