PRIDE Assigned Tags:Biomedical Dataset
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Human oral cancer brush biopsy iTRAQ
iTRAQ-based comparison of proteins derived from oral cells collected by brush biopsy. Protein abundance levels compared between oral pre-malignant cells, oral cancer cells and healthy normal cells, all collected from human patients. Two separate iTRAQ labeled biological replicate analyses were conducted.
Sample Processing Protocol
Cells were lysed, proteins digested with trypsin and iTRAQ labeled. Combined peptide mixtures were fractionated by high pH HPLC offline, and combined fractions were analyzed via LC-MS/MS on an Orbitrap Velos using HCD fragmentation.
Data Processing Protocol
Raw files were converted to mzXml using msconvert (distributed as part of ProteoWizard 1.6.1260). MS/MS spectra were searched against the Uniprot human database including scrambled sequences and common contaminant proteins (a total of 136,002 entries) using Sequest v27.0. Search parameters included a 1.6 amu (atomic mass units) precursor and 0.8 amu fragment mass tolerance, 2 missed cleavages, partial trypsin specificity, fixed modifications of carbamidomethylated cysteine, iTRAQ reagent modification at lysines and N-termini, and variable modification of methionine oxidation. Search results were filtered to 99% protein probability and 95% peptide probability in Scaffold (v3.3.1, Proteome Software), producing a false discovery rate of 1%. Proteins were quantified using customized software developed in-house call iQuant.
Yang Y, Rhodus NL, Ondrey FG, Wuertz BR, Chen X, Zhu Y, Griffin TJ. Quantitative proteomic analysis of oral brush biopsies identifies secretory leukocyte protease inhibitor as a promising, mechanism-based oral cancer biomarker. PLoS One. 2014 Apr 18;9(4):e95389. eCollection 2014 PubMed: 24748380