Comprehensive Protein Identification Protocol

The DIA data were analyzed with Spectronaut 5, a mass spectrometer vendor-independent software from Biognosys. The default settings were used for the Spectronaut search. Retention time prediction type was set to dynamic iRT (correction factor for window 1). Decoy generation was set to scrambled (no decoy limit). Interference correction on MS2 level was enabled. The false discovery rate (FDR) was set to 1% at peptide level. The DDA spectra were analyzed with the MaxQuant Version 1.4.1.2 analysis software using default settings with the following alterations (29 (link)). The minimal peptide length was set to 6. Search criteria included carbamidomethylation of cysteine as a fixed modification, oxidation of methionine and acetyl (protein N terminus) as variable modifications. The mass tolerance for the precursor was 4.5 ppm and for the fragment ions was 20 ppm. The DDA files were searched against the human UniProt fasta database (state 29.04.2013, 20,254 entries), the spike in proteins (12 entries), and the Biognosys iRT peptide sequences (11 entries). The identifications were filtered to satisfy FDR of 1% on peptide and protein level.

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Bruderer R., Bernhardt O.M., Gandhi T., Miladinović S.M., Cheng L.Y., Messner S., Ehrenberger T., Zanotelli V., Butscheid Y., Escher C., Vitek O., Rinner O, & Reiter L. (2015). Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues. Molecular & Cellular Proteomics : MCP, 14(5), 1400-1410.

Publication 2015

Cysteine Human Ions Methionine Peptide Protein Protein n Retention Spike proteins Tolerance

Corresponding Organization : Biognosys (Switzerland)

Other organizations : ETH Zurich, Purdue University West Lafayette

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Protocol cited in 88 other protocols

Variable analysis

independent variables

Spectronaut 5 search settings
MaxQuant Version 1.4.1.2 analysis settings

dependent variables

Peptide identifications
Protein identifications

control variables

Retention time prediction type (dynamic iRT)
Decoy generation (scrambled with no decoy limit)
Interference correction on MS2 level
False discovery rate (FDR) at 1% on peptide level
Minimal peptide length set to 6
Carbamidomethylation of cysteine as a fixed modification
Oxidation of methionine and acetyl (protein N terminus) as variable modifications
Mass tolerance for precursor at 4.5 ppm and for fragment ions at 20 ppm
Database search against human UniProt fasta, spike-in proteins, and Biognosys iRT peptide sequences
FDR filtering at 1% on peptide and protein level

positive controls

Biognosys iRT peptide sequences

negative controls

Not specified

Annotations

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