HIGH RESOLUTION MASS SPECTROMETRY WITH CHIP-BASED IONIZATION FOR THE ASSESSMENT OF NONCOVALENT INTERACTIONS OF PROTEINS WITH NORMAL BRAIN AND BRAIN TUMOR GANGLIOSIDES

Abstract

The noncovalent interactions between the amyloid beta (Aβ) and α-synuclein (Syn) proteins with native gangliosides extracted and purified from human brain and human glioma were studied using an analytical platform encompassing fully automated chip-nanoelectrospray (nanoESI) on a NanoMate robot coupled to a quadrupole time-of-flight (QTOF) mass spectrometer (MS). The novel interaction assays developed here for this purpose involved several stages: i) the incubation at 37°C under constant steering of the protein and gangliosides in 10 mM ammonium acetate buffer, pH 6.0, up to a concentration of 1 pmol μL-1 and 10 pmol μL-1, respectively; ii)collection of aliquots directly into the 96-well plate of the robot after 1, 5, 10, 15, 30, 60 and 180 min of incubation and iii) immediate submission of the reaction products to MS screening and structural analysis. Chip-nanoESI QTOF MS and CID MS/MS revealed the formation of the Aβ-GT1(d18:1/18:0) and Aβ-GT1 (t18:1/18:0) complexes as well as the preferential biding of Syn to GD1, GT1, GQ1 and GO1 species. CID MS/MS top-down fragmentation analysis demonstrated that the Aβ protein binds to a GT1b isomer type structure, characterized by Neu5Ac linkage to the external galactose and a disialo element Neu5Ac-Neu5Ac bound to the inner galactose of the molecule. Thus, by chip-MS and tandem MS experiments it was possible to deduce the structure of this non-covalent complex as: Aβ-GT1b
(d18:1/18:0). Similar results were obtained also for the Aβ complex formed with the gangliosides having trihydroxylated ceramide as well as the complexes of Syn with G1 class of gangliosides.