LC-MS/MS raw files were analyzed by MaxQuant (1.6.10.43) software for peptide/protein identification and quantification.24 (link) Uniprot Homo sapiens proteome database (Swiss-Prot, cononical) was used for protein identification (1% false discovery rate cutoff) with a fixed modification of cysteine carbamidomethylation and variable modifications of oxidation of methionine, acetylation of protein N-terminus, and biotin–phenol modification of tyrosine. PEAKS Studio X software was used to conduct a semiopen post-translational modification (PTM) search. The PEAKS PTM algorithm was used to search for 309 potential preset modifications. Additional modifications for oxidation at other amino acid residues were imported from the Unimod database.25 (link) Maxquant and PEAKS output files were analyzed in Excel and R for statistical analysis. Protein network analysis was conducted with STRING.26 (link) For data normalization to the most abundant biotinylated protein (PCCA), raw protein intensities from Maxquant output were normalized to the PCCA intensities followed by log2-transformation before statistical analysis. Raw proteomics data from this manuscript are available through the MassIVE repository27 (link) (Identifier: MSV000086260).
Protocol detail
Quantitative Proteomic Analysis of Biotinylated Proteins
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Acetylation
Amino acid
Biotin
Cysteine
Homo sapiens
Methionine
Ms ms
Pcca
Peptide
Phenol
Post translational modification
Protein
Protein n
Proteome
Tyrosine
Corresponding Organization : George Washington University
Other organizations : National Institute of Neurological Disorders and Stroke, University of Cambridge
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Variable analysis
independent variables
- Protein database used for identification (Uniprot Homo sapiens proteome database, Swiss-Prot, canonical)
- Modifications searched (cysteine carbamidomethylation, methionine oxidation, protein N-terminus acetylation, tyrosine biotin–phenol modification, additional modifications for oxidation at other amino acid residues)
- Peptide and protein identification
- Protein quantification
- False discovery rate cutoff (1%) for protein identification
- Positive control: Not specified
- Negative control: Not specified
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