LC-MS/MS assay for the investigation of acetylated Alpha-synuclein in serum from postmortem Alzheimer's disease pathology.

Alpha-synuclein (α-Syn), a neuronal protein, has been linked to the inflammation and development of neurodegenerative diseases. In a number of neurodegenerations, α-Syn has been investigated in the central nervous system and cerebrospinal fluid. However, there are few studies concerning the variations in peripheral α-Syn in postmortem Alzheimer's disease (AD) pathology. In this study, the quantitative procedure for the determination of peripheral acetylated α-Syn regarding N-terminal amino acid's site (α-Syn1-6; MDVFMK and Ac-α-Syn1-6; Ac-MDVFMK) was developed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and tryptic digestion without antibody. Serum samples were selected from postmortem specimens based on autopsy pathological examination of AD remark. The LC-MS/MS assay with ACQUITY UPLC BEH C18 column was applied on the basis of electrospray positive ionization. When subjected to N-terminal α-Syn peptides using MonoSpin Typsin HP preparation, doubly- and singly-charged α-Syn1-6 and Ac-α-Syn1-6 ions were observed at m/z 386 > 104 and m/z 813 > 72, respectively, which correspond to quantitative profiling with internal standards. In the calibration, the range of 10-1000 nmol/L showed r2 = 0.999 and recovery from 86.0% to 115.0% (RSD < 9.0%). Using this procedure, peripheral α-Syn1-6 from serum samples could not be detected. On the other hand, Ac-α-Syn1-6 levels were measured from 106.9 to 319.8 nmol/L (AD; n = 10) and 147.1-292.0 nmol/L (control; n = 10) with an insignificant difference. From these preliminary results, individual Ac-α-Syn levels in serum were inferred with nonspecific biomarker regarding to AD pathology.