Dataset Information

Protein Expression Profile of PBMC from CA and OP patient samples

ABSTRACT: 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.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Minlin Ou

PROVIDER: PXD000346 | Pride | 2013-10-11

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 37

Similar Datasets

Project description:Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterised by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesise that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21. Mass spectra were analyzed using Mascot (version 2.2, Matrix Science), executing a spectral search against a concatenated International Protein Index (IPI) human protein database (version 3.54 containing 39,925 entries) and a decoy database. Parameters included: trypsin enzyme specificity, SILAC double measurements of Lys6 and Arg8, 1 missed cleavage, minimum peptide length of 7 amino acids, minimum of 1 unique peptide, top 6 MS/MS peaks per 100 Da, peptide mass tolerance of 20 ppm for precursor ion and MS/MS tolerance of 0.5 Da. Oxidation of methionine and N-terminal protein acetylation for variable modifications and cysteine caramidomethylation for fixed modification. All entries were filtered using a false positive rate of 1% both at the peptide and protein levels, and false positives were removed. Quantification via normalised H/L ratios was based on minimum of 3 peptide ratio counts. Protein group entries with a normalised ratio significance B score of =< 0.05 or significance A score of =< 0.05 were retained for further analysis.

			Action	DRS
	Mascot.dat	Other
	group1.wiff	Wiff
	group1.wiff.mtd	Wiff
	group1.wiff.scan	Wiff
	group10.wiff	Wiff

Dataset Information

Protein Expression Profile of PBMC from CA and OP patient samples

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets