Computational protocol: NDUFAF7 Methylates Arginine 85 in the NDUFS2 Subunit of Human Complex I*

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Protocol publication

[…] The 66 known and 142 putative human methyltransferases () were examined for the presence of N-terminal mitochondrial targeting sequences with the programs MitoProt, iPSORT, and TargetP (–). [...] The effect of the suppression of expression of NDUFAF7 on the methylation status of NDUFS2 was studied by analyzing the two tryptic peptides (residues 75–89 and 76–89) containing the methylated arginine 85. Crude mitoplast proteins were reduced with 5 mm Tris (2-carboxyethyl)-phosphine, pH 7.0, alkylated with 15 mm iodoacetamide in SDS gel sample buffer at pH 8.0, and fractionated on Novex Tris-glycine 10–20% (Invitrogen) acrylamide gradient gels. Proteins were identified by tryptic mass mapping of Coomassie Blue-stained bands or sections of gel () in a MALDI-TOF-TOF mass spectrometer (model 4800; AB-Sciex, Warrington, UK) with α-cyano-4-hydroxycinnamic acid as matrix. CID was performed with air. Peptides were fractionated also by reverse phase nanochromatography with a Proxeon Easy-nLC instrument (Thermo Fisher, Hemel Hempstead, UK). The C18 column (75-μm inner diameter × 100 mm; Nanoseparations, Nieuwkoop, The Netherlands) was eluted with an acetonitrile gradient in 0.1% (v/v) formic acid at 250 nl/min and was coupled directly to an LTQ OrbiTrap XL-ETD (electron transfer dissociation) mass spectrometer (Thermo Fisher). Peptides were fragmented by CID with nitrogen. Proteins were identified from peptide mass and fragmentation data by interrogation of NCBInr protein sequence databases with Proteome Discoverer 1.3 (Thermo Fisher) and the Mascot search engine (). The relative abundance of two methylated tryptic peptides (residues 76–89 and 75–89 of subunit NDUFS2; CamDPHIGLLHRGTEK and KCamDPHIGLLHRGTEK), plus one nonmethylated version (residues 75–85, KCamDPHIGLLHR) found only in cells where expression of NDUFAF7 had been suppressed, were estimated from the peak areas of Gaussian smoothed extracted ion chromatograms obtained with Xcalibur (Thermo Fisher) using an m/z tolerance of 5 ppm. Monoisotopic m/z values used are 830.9336 (M + 2H)2+, 554.2916 (M + 3H)3+, and 894.9810 (M + 2H)2+, 596.9899 (M + 3H)3+ for the two methylated peptides, respectively, and 673.3620 (M + 2H)2+ and 449.2439 (M + 3H)3+ for the nonmethylated peptide. The two methylated tryptic peptides were identified by Proteome Discoverer from the fragmentation spectrum produced by CID. To aid the identification of the unmethylated peptide representing residues 75–85 of subunit NDUFS2, a synthetic version was obtained from Cambridge Research Biochemicals (Billingham, UK). […]

Pipeline specifications

Software tools MITOPROT, iPSORT, TargetP, Proteome Discoverer, Mascot Server
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Homo sapiens
Chemicals NAD, S-Adenosylmethionine, Ubiquinone