Computational protocol: Bottom-Up Proteomics of Escherichia coli Using Dynamic pH Junction Preconcentration and Capillary Zone Electrophoresis-ElectrosprayIonization-Tandem Mass Spectrometry

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Protocol publication

[…] Database searching of acquired raw files was performed in Proteome Discoverer 1.3 with the Mascot search engine (version 2.2) against ipi.bovin.v3.68.fasta (for BSA) and the NCBI E. coli DH1 database (for E. coli). Database searching of the reversed database was also performed in order to evaluate the false discovery rate. The database searching parameters included full tryptic digestion and allowed up to two missed cleavages, precursor mass tolerance 2 Da, and fragment mass tolerance 1 Da. Carbamidomethylation (C) was set as fixed modifications. Oxidation (M) was set as variable modification.Percolator software (version 1.17) integrated in Proteome Discoverer 1.3 was used to evaluate the database search results. Peptide confidence value as high was used to filter the peptide identifications, and the corresponding false discovery rate at the peptide level was less than 1%. On the protein level, the protein grouping was enabled.Data were imported into Matlab. Selected ion electropherograms were generated with a 10 ppm mass window. An unsupervised least-squares routine (“fit”) was used to fit a Gaussian function to selected ion electropherograms. The peak area was estimated as the product of peak amplitude and width. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server
Application MS-based untargeted proteomics
Organisms Escherichia coli