Human bone protein and peptide output from PEAKS software
Rib bone (< 10 g) were collected post-mortem from five male, and five female deceased subjects. Soft tissue was removed from each rib using a scalpel and a 20 mg block of cortical bone was removed. The sample processing workflow on samples included protein extraction and peptide digestion using a modified acidic demineralization protocol. Data acquisition was performed using Thermo Scientific Q Exactive Plus Orbitrap mass spectrometer coupled to an Easy-nLC 1000 liquid chromatograph (Thermo Scientific; Waltham, MA; USA). Mass spectral data were obtained using a “top-10” data-dependent collection strategy in which an initial MS scan over the range of m/z 380-1800 and a resolution of 70,000 was used to select 10 precursor ions for subsequent MSMS scans. RAW data was exported and analyzed in parallel using two protein identification software programs: PEAKS 7.5 (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada).RAW datafiles were directly imported into software. Oxidation of methionine, carbamidomethylation of cysteine, deamidation of asparagine and glutamine, and hydroxylation of proline were included in the search settings as partial post-translational modifications. Precursor mass error of 15 ppm using monoisotopic mass was used for parent ion identifications and 0.05 Da for fragment ions masses. Protein identifications (IDs) were filtered by a 1% FDR.