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Oligosaccharide Assembly01:24

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Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may...
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Top/Middle-Down Characterization of α-Synuclein Glycoforms.

Samuel A Miller1, Kevin Jeanne Dit Fouque1, Eldon R Hard2

  • 1Department of Chemistry and Biochemistry and Biomolecular Sciences Institute, Florida International University, Miami, Florida 33199, United States.

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|December 4, 2023
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Summary

This study introduces a novel mass spectrometry method for identifying specific O-linked N-acetyl-glucosamine (O-GlcNAc) modifications on alpha-synuclein. This advancement aids in understanding neurodegenerative diseases like Parkinson's by characterizing alpha-synuclein glycoforms.

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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.