Computational protocol: Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

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Protocol publication

[…] Protein sequences were obtained by BLAST using NCU09018 (QC-1) and NCU11249 (QC-2) as protein search queries. Sequences were aligned using MAFFT version 7 (). The alignment was used to construct a maximum likelihood phylogeny using RAxML (). FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) was used for visualization. [...] Trypsin-digested protein samples were analyzed using a Thermo-Dionex UltiMate3000 RSLCnano liquid chromatograph that was connected in-line with an LTQ-Orbitrap-XL mass spectrometer equipped with a nanoelectrospray ionization (nanoESI) source (Thermo Fisher Scientific, Waltham, MA). The LC was equipped with a C18 analytical column (Acclaim PepMap 100; Thermo) (150-mm length, 0.075-mm inner diameter, 3-µm particles, 100-Å pores) and a 1-µl sample loop. Acetonitrile (Fisher Optima grade, 99.9%), formic acid (1-ml ampules, 99+%; Thermo Pierce), and water purified to a resistivity of 18.2 MΩ ⋅ cm (at 25°C) using a Milli-Q Gradient UltraPure water purification system (Millipore, Billerica, MA) were used to prepare mobile phase solvents. Solvent A was 99.9% water–0.1% formic acid, and solvent B was 99.9% acetonitrile–0.1% formic acid (vol/vol). The elution program consisted of isocratic flow with 2% solvent B for 4 min, a linear gradient to 30% solvent B over 38 min, isocratic flow with 95% solvent B for 6 min, and isocratic flow with 2% solvent B for 12 min, at a flow rate of 300 nl/min.Full-scan mass spectra were acquired in the positive-ion mode over the m/z range from 350 to 1,800 using the Orbitrap mass analyzer, in profile format, with a mass resolution setting of 30,000 (at m/z = 400, measured at full width at half-maximum peak height [FWHM]). In the data-dependent mode, the eight most intense ions exceeding an intensity threshold of 50,000 counts were selected from each full-scan mass spectrum for tandem mass spectrometry (MS/MS) analysis using collision-induced dissociation (CID). MS/MS spectra were acquired using the linear ion trap, in centroid format, with the following parameters: isolation width of 3 m/z units, normalized collision energy of 30%, default charge state of 3+, activation Q = 0.25, and activation time of 30 ms. Real-time charge state screening was enabled to exclude unassigned and 1+ charge states from MS/MS analysis. Real-time dynamic exclusion was enabled to preclude reselection of previously analyzed precursor ions, with the following parameters: repeat count of 3, repeat duration of 10 s, exclusion list size of 500, exclusion duration of 90 s, and exclusion mass width of 20 ppm. Data acquisition was controlled using Xcalibur software (version 2.0.7; Thermo). Raw data were searched against the amino acid sequence of N. crassa CBH-1 (NCU07340) using Proteome Discoverer software (version 1.3, SEQUEST algorithm; Thermo) for tryptic peptides with up to three missed cleavages, and carbamidomethylcysteine, methionine sulfoxide, and N-terminal pyroglutamate as variable posttranslational modifications. Peptide identifications were validated by manual inspection of the MS/MS spectra, i.e., to check for the presence of y-type and b-type fragment ionsS1 that identify the peptide sequences. Data acquisition and integration of extracted ion chromatograms of peptide ions were performed using Xcalibur software (version 2.0.7; Thermo). MS/MS spectra are annotated using the nomenclature of Roepstorff and Fohlman (). […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Organisms Neurospora crassa, Homo sapiens
Chemicals Amino Acids, Carbohydrates, Pyrrolidonecarboxylic Acid, Glutamic Acid