Top/Middle-Down Characterization of α-Synuclein Glycoforms

Area of Science:

• Biochemistry
• Neuroscience
• Analytical Chemistry

Background:

• Alpha-synuclein is crucial in neurodegenerative diseases, with O-linked N-acetyl-glucosamine (O-GlcNAc) modifications influencing its aggregation and toxicity.
• Current methods lack site-specific screening tools for O-GlcNAc alpha-synuclein, hindering therapeutic development.

Purpose of the Study:

• To develop and validate a mass spectrometry-based approach for characterizing alpha-synuclein positional glycoforms.
• To enable high-throughput screening of O-GlcNAc modifications for better therapeutic strategies in synucleinopathies.

Main Methods:

• Utilized trapped ion mobility spectrometry (TIMS) coupled with mass spectrometry (MS) employing electron capture dissociation (ECD) and 213 nm ultraviolet photodissociation (UVPD).
• Applied top-down and middle-down MS/MS approaches, including proteolytic digestion and TIMS-q-ECD-TOF MS/MS, for glycoform characterization.
• Analyzed alpha-synuclein modified with single O-GlcNAc at T72, T75, T81, and S87 positions.

Main Results:

• Top-down UVPD and ECD MS/MS provided specific product ions for each glycoform, achieving high sequence coverage (up to ~82%).
• TIMS-MS revealed structural heterogeneity and distinct mobility profiles for glycoforms, though separation was partial.
• Middle-down approach successfully separated and identified positional glycoforms with high confidence (~80% sequence coverage).

Conclusions:

• The developed TIMS-MS/MS method, incorporating ECD and UVPD, enables precise characterization of alpha-synuclein positional glycoforms.
• This technique offers a powerful tool for high-throughput screening, advancing structural mass spectrometry for synucleinopathy research.
• The findings facilitate a deeper understanding of O-GlcNAc's role in alpha-synuclein function and disease pathogenesis.