Computational protocol: Selenium-Based S-Adenosylmethionine Analog Reveals the Mammalian Seven-Beta-Strand Methyltransferase METTL10 to Be an EF1A1 Lysine Methyltransferase

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Protocol publication

[…] Acetonitrile (1/10 volume) and DTT (20 mM) were added to the protein-bound Dynabeads in 100 mM ABC buffer, and the mixture was incubated for 30 min at 56°C. Then, iodoacetamide (IAA) was added and the mixture was incubated for 30 min at 37°C in the dark. The protein samples were then digested with 0.5 µg trypsin (Promega). The trypsinized protein fragments were applied to a liquid chromatograph (EASY-nLC 1000; Thermo Fisher Scientific, Odense, Denmark) coupled to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Inc., San Jose, CA, USA) with a nanospray ion source in positive mode. The peptides derived from the protein fragments were separated on a NANO-HPLC capillary column C18 (0.075-mm inner diameter ×150 mm length, 3 mm particle size; Nikkyo Technos, Tokyo, Japan). Mobile phase “A” was comprised of water with 0.1% formic acid, and mobile phase “B” was comprised of acetonitrile with 0.1% formic acid. Two different slopes were used for a 60 min gradient at a flow rate of 300 nL/min: 5%–35% B in 48 min and then 35%–65% B in 12 min. The mass spectrometer was operated in the top-10 data-dependent scan mode. The parameters of the mass spectrometer were as follows: spray voltage, 2.3 kV; capillary temperature, 275°C; mass-to-charge ratio, 350–1800; normalized collision energy, 28%. Raw data was acquired with the Xcalibur software (Thermo Fisher Scientific). The MS and MS/MS data were searched against the Swiss-Prot database using Proteome Discoverer 1.4 (Thermo Fisher Scientific) with the MASCOT search engine software, version 2.4.1 (Matrix Science, London, United Kingdom). The search parameters were as follows: enzyme, trypsin; static modifications, carbamidomethyl (Cys); dynamic modifications, oxidation (Met); precursor mass tolerance, ±6 ppm; fragment mass tolerance, ±20 mDa; maximum missed cleavages, 1. The proteins were considered identified when their false discovery rates (FDR) were less than 1%. The obtained data were further filtered as follows: peptide rank, 1 and peptides per proteins, minimal 3. Nonlabel quantification was performed by Top3-TIC method with Proteome Discoverer. Briefly, peptide areas of three most abundant peptides were used for quantification of protein amounts. For substrate identification, proteins with at least a 2-fold increase in three independent experiments were defined as positive hit proteins. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server
Databases UniProt
Application MS-based untargeted proteomics
Organisms Homo sapiens, Saccharomyces cerevisiae
Chemicals S-Adenosylmethionine, Selenium