Computational protocol: Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics

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Protocol publication

[…] Spectra were searched in PEAKS (version 7.5; Bioinformatics Solutions Inc.) using a precursor mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.2 Da. Two missed cleavages were allowed, as well as nonspecific cleavage at one end of the peptide. The following post translational modifications were allowed: fixed—carbamidomethylation [C]; variable—oxidation [M], oxidation or hydroxylation [RYFPNKD], [G]@C terminal, pyro-glu from Q, and deamidation [NQ]. Spectra were searched against the UniProt_chicken database, and PEAKS PTM and SPIDER were enabled to account for unspecified post-translational modifications (PTMs) or mutations, respectively. Results were filtered using ≤1% FDR for peptide spectral matches (PSMs), and either a protein score of −log10p ≥ 20, or ≤1% FDR (whichever was more stringent) plus at least 1 unique peptide for proteins. Effective FDR values and associated expect score cut-offs for each file searched, as well as the number of MS and MS/MS scans performed, is provided in . Search results were analyzed by hand: peptides corresponding to common lab contaminants (e.g., keratin) were eliminated from consideration, and duplicate matches of one spectrum to multiple protein accession IDs were reduced. Additionally, proteins identified by only 1 peptide in 1 injection were eliminated (23 proteins), and only those found in multiple injections, or as multiple peptides from a single injection, were used for comparison. Finally, data from duplicate trials of the same fractions were combined to account for variation between trials. […]

Pipeline specifications

Software tools PeaksPTM, SPIDER
Application MS-based untargeted proteomics