Computational protocol: A novel mass spectrometric strategy “BEMAP” reveals Extensive O-linked protein glycosylation in Enterotoxigenic Escherichia coli

Similar protocols

Protocol publication

[…] Raw data generated on the LTQ Orbitrap Velos or Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) were processed with Proteome Discoverer (Version 1.4.1.14, Thermo Fisher Scientific) and subjected to database searching using an in-house Mascot server (Version 2.2.04, Matrix Science Ltd., London, UK). Database searches were performed with the following parameters: Database: annotated E. coli proteomes from ETEC H10407, AIEC LF82, NMEC IHE3034 and commensal MG1655; Trypsin as the enzyme allowing a maximum of one missed cleavages sites; Carbomidomethylation of Cys as fixed modification; Deamidation of Asn and Gln; Oxidation of Met allowed as variable modification; When required BEMAP mass tag 2-AEP ((C(2) H(6) N O(2) P) on Ser/Thr or Heptose on Ser/Thr or GlcNAc on Ser/Thr was set as variable modification. Precursor and fragment mass tolerance were set to 10 ppm and 0.05 Da, respectively. Precursor mass range set from 350 Da to 7,000 Da. False discovery rate was set to 1% at peptide level using the Percolator algorithm.Our in house developed tool Peptide Finder (accessible at www.microbiology.sdu.dk) was used to extract and compile a list of uniquely modified peptides using a ProteomeDiscover output text file as template. Similarly, our in house developed script MotifX aligner (accessible at www.microbiology.sdu.dk) was used to extract and centre Ser or Thr modified residues with nine flanking residues; Number of flanking amino acids is user defined. ProteinCenter (Thermo Scientific, Germany) was used to assign Gene Ontology terms to all identified proteins. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server, ProteinCenter
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Escherichia coli, Escherichia coli K-12, Bacteria
Diseases Genetic Diseases, X-Linked
Chemicals Carbohydrates, Oxygen