Computational protocol: Tyrosine glycosylation of Rho by Yersinia toxin impairs blastomere cell behaviour in zebrafish embryos

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Protocol publication

[…] Glycosylation of RhoA, Rac1 and Cdc42 (5 μg each) was performed with Afp18G (100 nM) in the presence of UDP-GlcNAc (1 mM) for 30 min at 30 °C in glycosylation buffer. After SDS–PAGE, excised gel bands were destained with 30% acetonitrile (ACN), shrunk with 100% ACN and dried in a vacuum concentrator. Subsequently, protein digestion with thermolysin was performed overnight at 37 °C in 0.1 M NH4HCO3 (pH 8). Approximately 0.1 μg of protease per gel band was used. Peptides were extracted from the gel matrix with 5% formic acid and subsequently analysed by LC–MS on a Q-TOF mass spectrometer (Agilent 6520, Agilent Technologies) coupled to a 1200 Agilent nanoflow system via a HPLC-Chip cube electrospray ionization interface. Peptides were separated on a HPLC-Chip with an analytical column of 75 μm i.d. and 150 mm length and a 40-nl trap column, both packed with Zorbax 300SB C-18 (5 μm particle size). Starting with 3% ACN, a linear gradient with 1% per min at a flow rate of 300 nl min−1 was applied. The Q-TOF instrument was operated in the 2-GHz extended dynamic range mode and MS spectra were acquired in the mass-to-charge (m/z) range between 50 and 3,000. For MS–MS analyses, the instrument was operated in the data-dependent acquisition mode. After a survey MS scan (four spectra s−1), a maximum of three multiple charged peptides were consecutively selected for MS–MS experiments (two spectra s−1). Internal mass calibration was applied. Mascot Distiller 2.4.2 was used for raw data processing and for generating peak lists, essentially with standard settings for the Agilent Q-TOF instrument. Mascot Server 2.3 was used for searches in the SwissProt protein database with the following parameters—peptide mass tolerance, 50 p.p.m.; MS–MS mass tolerance, 0.05 Da; enzyme, no specificity; variable modifications, Carbamidomethyl (C), Gln-> pyroGlu (N-term. Q), oxidation (M) and HexNAc (STY). [...] Diffraction data were collected at 100 K with a Rigaku M007HF X-ray generator and a Saturn 944HG+ CCD detector. The wavelength was 1.54 Å. Data were processed with HKL3000R. The initial phases were calculated by molecular replacement using Molrep from the CCP4 software suite with RhoA (PDB code 1pdf) as a search model using the reflections from 50 to 3.5 Å. The structure was further refined by rigid body and iterative restraint refinement with the software Refmac5 (ref. ) and model building in COOT. Structural data are summarized in . The electron density of the GlcNAc moiety, GDP and a sulfate were clearly observed in the unbiased Fo−Fc map, but no Mg2+ ion was found. Electron density of the side chains from amino acid Glu32, Arg128, and Glu142 were poorly defined; therefore, amino acids were modelled as alanines. A crystallographic information file with the refinement restraints of the alpha-GlcNAc-Tyr bond was created in JLigand. Structure images were prepared using PyMOL (http://www.pymol.org/). [...] Analysis of variance was performed using GraphPad PRISM. Kruskal–Wallis and Mann–Whitney U-tests were applied. Student's t-test was applied for pairwise statistical comparison. Fisher's exact test was applied for statistical analysis of the data set in , using VassarStats (http://vassarstats.net/). […]

Pipeline specifications

Software tools Mascot Distiller, Mascot Server, Molrep, CCP4, REFMAC5, Coot, PyMOL, VassarStats
Applications Miscellaneous, MS-based untargeted proteomics, Protein structure analysis
Organisms Danio rerio, Homo sapiens
Diseases Enterocolitis, Zoonoses
Chemicals Guanosine Diphosphate, Guanosine Triphosphate, Tyrosine