Identification of the native Torpedo californica nicotinic acetylcholine receptor's glycan composition after a multi-step sequential purification method using MALDI-ToF MS

Proteomics. 2024 Jan;24(1-2):e2300151. doi: 10.1002/pmic.202300151. Epub 2023 Oct 30.

Abstract

The Cys-loop pentameric ligand-gated ion channels comprise a dynamic group of proteins that have been extensively studied for decades, yielding a wealth of findings at both the structural and functional levels. The nicotinic acetylcholine receptor (nAChR) is no exception, as it is part of this large protein family involved in proper organismal function. Our efforts have successfully produced a highly pure nAChR in detergent complex (nAChR-DC), enabling more robust studies to be conducted on it, including beginning to experiment with high-throughput crystallization. Our homogeneous product has been identified and extensively characterized with 100% identity using Nano Lc MS/MS and MALDI ToF/ToF for each nAChR subunit. Additionally, the N-linked glycans in the Torpedo californica-nAChR (Tc-nAChR) subunits have been identified. To study this, the Tc-nAChR subunits were digested with PNGase F and the released glycans were analyzed by MALDI-ToF. The MS results showed the presence of high-mannose N-glycan in all native Tc-nAChR subunits. Specifically, the oligommanose population Man8-9GlcNac2 with peaks at m/z 1742 and 1904 ([M + Na]+ ions) were observed.

Keywords: Lc MS/MS; MALDI ToF/ToF; N-linked glycans; Torpedo californica; nAChR-DC.

MeSH terms

  • Acetylcholine / metabolism
  • Animals
  • Nicotine*
  • Receptors, Nicotinic* / chemistry
  • Receptors, Nicotinic* / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Tandem Mass Spectrometry
  • Torpedo / metabolism

Substances

  • Nicotine
  • Acetylcholine
  • Receptors, Nicotinic