Protein Expression Profile of PBMC from CA and OP patient samples

To identify and quantify protein profiles from peripheral blood mononuclear cells (PBMC) of osteopetrosis patients with isobaric Tagging for Relative and Absolute protein Quantification (iTRAQ) based proteomic technology and to find differentially expressed proteins in osteopetrosis. Total protein was extracted from PBMC samples of 6 osteopetrosis patients and 9 health donors. For the osteopetrosis group and the control group, aliquots of protein from each subject were pooled respectively. One hundred micrograms of protein from each corresponding group pool protein was digested with trypsin, and labeled according to the iTRAQ protocol. The labeled digests were then combined into one sample mixture and subjected to LC-MS/MS analysis. Identification and relative quantification of proteins based on the ratio of peak areas from MS/MS spectra, and the m/z of 114 (osteopetrosis) and 115 (control) were involved in this research. The MS data were processed using Proteome Discoverer Software (Version 1.2.0.208) to generate the peak list. Mascot 2.3.02 was used for the protein identification, and the parameters as follows: Type of search: MS/MS Ion search; Enzyme: Trypsin; Fragment mass tolerance: ±0.1 Da; Mass values: Monoisotopic; Variable modifications: Gln->pyro-Glu (N-term Q), Oxidation (M), iTRAQ8plex(Y); Peptide mass tolerance: 0.05 Da; Instrument type: Defult; Max missed cleavages: 1; Fixed modifications: arbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K); Protein Mass: Unrestricted; Other parameters: default; Database, IPI_human v3.87 (91464 sequences). The search results were further filtered with the parameters as follows: for protein identification, significance threshold: P<0.05 (with 95% confidence), ion score or expected cutoff < 0.05 (with 95% confidence); for protein quantitation, protein ratio type: weighted; minimum precursor charge: 1; minimum peptides: 2 (unique peptides); median intensities: normalization (removed outliers automatically). Proteins with 1.5 fold or more change between osteopetrosis group and the control group were determined as differentially expressed proteins.