N-terminally acetylated full-length human α-synuclein was the major isoform detected in a ASOTg cortex mitochondrial fraction and in human brain by top-down high-resolution mass spectrometry.

<p>A. Total ion chromatogram is shown for the primary separation and the ion-isolation mass spectrum (m/z 967.76) of a candidate full-length α-synuclein of mass 14503.1 Da found in the 37-minute fraction for a ASOTg cortex mitochondrial fraction (500 µg). B. Static nano-electrospray and MSMS displays peaks of various intensities for the average mass spectrum in the same 37-minute fraction (arrow, unmodified human α-synuclein sequenced in panel C). No masses corresponded to known PTMs for human α-synuclein, e,g, nitration, phosphorylation or ubiquitination. C. Hybrid linear ion-trap FT-MS (LTQ-FT) generates high resolution map of product ions formed upon collisionally activated dissociation (CAD) of the m/z 967 precursor, matched at 10 ppm tolerance for confident assignment of the primary amino acid sequence of full-length human α-synuclein from ASOTg mouse (upper panel). Monoisotopic mass of human α-synuclein from ASOTg mouse was 14493.2591 Da (mean of 4 measurements on 4 different ions). A protein of similar mass was recovered from resected human brain and CAD of the m/z 1209 precursor gave a broadly similar product-ion map (lower panel). Monoisotopic mass of human α-synuclein from human brain was 14493.2592 Da (mean of 2 measurements on 2 different ions). The probability that either species analyzed was incorrectly assigned was calculated to be 9.9×10⁻³¹ (ASOTg) and 4.4×10⁻⁴³ (human brain) using the ProsightPC algorithm at a tolerance of 10 ppm. Note that the top-down approach yields several product ions in the C-terminal region that was poorly covered in the bottom-up experiment (Fig. 6). The species analyzed is shown with N-terminal acetylation of starting methionine (in red) yielding agreement of measured and calculated masses within 10 ppm.