title: A technically detailed and pragmatic protocol for quantitative serum proteomics using iTRAQ.
abstract: Blood is recognised as a highly important source of disease-related biomarkers, and proteomic approaches for identifying novel blood-borne biomarkers are in demand. The complexity and dynamic protein concentration range of plasma/serum however complicates the analysis process. A number of strategies for simplification of blood prior to proteomic analysis have been developed. In addition, methods for quantifying the levels of proteins in samples, such as isobaric tags for relative and absolute quantification (iTRAQ) are emerging. However, the successful application of these procedures is not always straightforward and technical hurdles must be overcome. Here we provide a technically detailed working protocol for iTRAQ-based quantification of serum proteins following immunodepletion of high abundance proteins. To improve the number of proteins identified and quantified we have introduced several modifications to the standard iTRAQ protocol. We report identifications of 217 proteins (5773 peptides) with a false discovery rate of 1% or 254 proteins with 95% confidence, respectively. Relative quantification data were obtained for 234 (95% confidence) serum proteins, including species present in the concentration range of tissue leakage factors. The samples described here relate to pancreatic cancer; however the protocol can be applied to serum from other control or disease types.
authors: Royal Liverpool University Hospital, University of Liverpool, United Kingdom.
authors: Sarah
authors: S
authors: Tonack
authors: Mark
authors: M
authors: Aspinall-O'Dea
authors: Rosalind E
authors: RE
authors: Jenkins
authors: Victoria
authors: V
authors: Elliot
authors: Seonaid
authors: S
authors: Murray
authors: Catherine S
authors: CS
authors: Lane
authors: Neil R
authors: NR
authors: Kitteringham
authors: John P
authors: JP
authors: Neoptolemos
authors: Eithne
authors: E
authors: Costello
mesh_terms: D015415:Biomarkers / Q000097:blood; D014408:Biomarkers, Tumor / Q000097:blood; D001798:Blood Proteins / Q000032:analysis*; D006801:Humans; D009363:Neoplasm Proteins / Q000097:blood; D010190:Pancreatic Neoplasms / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012107:Research Design
chemical_list: D015415:Biomarkers; D014408:Biomarkers, Tumor; D001798:Blood Proteins; D009363:Neoplasm Proteins