Dataset Advancement: Glycan Composition and Peptide Mass Fingerprinting of the Native Torpedo californica Nicotinic Acetylcholine Receptor after a Multi-Step Sequential Purification Method

Description

The study described in this dataset focuses on the characterization of the nicotinic acetylcholine receptor (nAChR), a member of the Cys-loop pentameric ligand-gated ion channels that play critical roles in organismal function. Our research efforts have resulted in the successful production of a highly pure nAChR, which has allowed for more robust studies to be conducted, including high-throughput crystallization experiments. Using advanced technologies such as Nano Lc MS/MS and MALDI ToF/ToF, we have identified and extensively characterized each nAChR subunit, achieving a 100% identity. Furthermore, we have investigated the N-linked glycans in the Torpedo californica-nAChR (Tc-nAChR) subunits. By digesting the Tc-nAChR subunits with PNGase F and analyzing the released glycans with MALDI-ToF, we identified the presence of high-mannose N-glycan in all native Tc-nAChR subunits. Specifically, we observed the oligommanose population Man8-9GlcNac2 with peaks at m/z 1742 and 1904 ([M + Na]+ ions). Overall, this study emphasizes the importance of continued research efforts aimed at understanding the complex biology of the Cys-loop pentameric ligand-gated ion channels and their involvement in organismal function and disease. The insights gained from such studies could have far-reaching implications for the development of new therapeutic strategies to combat a wide range of health issues.

Steps to reproduce

1.1 MALDI ToF system and analysis for the native-nAChR peptide mass fingerprinting The native nAChR was extracted from the Tc electroplax tissue obtained from Aquatic Research Consultants, San Pedro, CA, USA, according to the protocols described by Maldonado-Hernández[1]. The in-gel tryptic digestion was performed by following the method of Shevchenko with modifications[2]. MALDI ToF analysis was performed on a 4800 Plus MALDI ToF Analyzer (SCIEX, MA, USA) and mass spectra were acquired in a reflector positive mode. Peptides were analyzed using 12 mg of 4-Chloro-α-cyanocinnnamic acid dissolved in 70% acetonitrile (vol/vol). Protein identification was achieved by searching the NCBIprot protein database using the Mascot search engine (Version 2.3.01). For coverage of sequence analysis, we used mMass Open-Source Mass Spectrometry Tool (http://www.mmass.org). 1.2 Lc-MS/MS system and analysis for the native-nAChR peptide mass fingerprinting Peptide purification and concentration were performed by reversed phase chromatography. The separation was obtained using a total gradient time of 69 minutes, as follows: of 5-40% of 0.1% of formic acid in 80% acetonitrile (Buffer B) for 45 minutes, 40-95% of Buffer B for 6 minutes, 5-95% Buffer B for an additional 12 minutes, and 95-5% for 6 minutes. Flow rate is set at 300 nL/min, and the injection volume is of 2 μL per sample. The Q-Exactive Plus is operated in positive polarity mode and a data-dependent mode. 1.3 The in-gel N-glycans release the native-nAChR The in-gel N-glycans release was performed according to Harvey, D. J. 2009 with the following modification: The gel bands corresponding to the nAChR subunits were excised and destain with 25 mM ammonium bicarbonate and 50% acetonitrile at 37°C for 30 minutes. Afterwards, protein disulfide bond reduction was performed by 20 minutes incubation at 37°C with 10 mM DTT in 50 mM ammonium bicarbonate. Then, the cysteine alkylation step was carried out in the dark for 45 minutes by adding 55 mM iodoacetamide in 50 mM ammonium bicarbonate at room temperature. Subsequently, N-glycans were released from 70 µg of the reduced and alkylated nAChR subunit by an overnight incubation at 37°C with 10 mU PNGase F. MS spectra for glycopeptides were analyzed in the linear positive ion mode using THAP matrix. Reference: 1. Maldonado-Hernández R, Quesada O, Colón-Sáez JO, Lasalde-Dominicci JA (2020) Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies. Analytical Biochemistry: 113887. doi: 10.1016/j.ab.2020.113887 2. Shevchenko A, Tomas H, Havli J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature protocols 1: 2856-2860. doi: 10.1038/nprot.2006.468 3. Harvey DJ (2009) In-gel Enzymatic Release of N-glycansThe Protein Protocols Handbook. Springer, pp. 1363-1370

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