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iTRAQ‐based quantitative analysis of protein mixtures with large fold change and dynamic range
Author(s) -
CasadoVela Juan,
MartínezEsteso María José,
Rodriguez Eva,
Borrás Eva,
Elortza Felix,
BruMartínez Roque
Publication year - 2010
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200900509
Subject(s) - fold (higher order function) , chemistry , fold change , computational biology , chromatography , biochemistry , biology , computer science , gene , downregulation and upregulation , programming language
Quantitation of changes in protein abundance is key to understanding the alterations that biological systems undergo and to discovering novel biomarkers. Currently, HPLC‐MS/MS can be used to quantify changes in protein expression levels [Ong, S. E. and Mann, M., Nat. Chem. Biol. 2005, 1 , 252–262]. Nevertheless, quantitative analysis of protein mixtures by HPLC‐MS/MS is still hampered by the wide range of protein expression levels, the high dynamic range of protein concentrations and the lack of reliable quantitation algorithms [D'Ascenzo, M., et al. Brief. Funct. Genomic. Proteomic. 2008, 7 , 127–135; Lin, W. T., et al. , J. Proteome Res. 2006, 5 , 2328–2338; Matthiesen, R., et al. J. Proteome Res. 2005, 4 , 2338–2347; Yu, C. Y., et al. Nucleic Acids Res. 2007, 35 , W707–W712]. In this context, we describe two different samples (4‐protmix and 8‐protmix) suitable for relative protein quantitation using iTRAQ. Using the 4‐protmix, relative protein changes of up to 24‐fold were measured. The 8‐protmix allowed the quantitation of the relative protein changes in a mixture of proteins within the range of two orders of magnitude in concentration and ten‐fold differences in relative abundance. We propose that the two samples are suited to test the iTRAQ quantitative proteomic workflow. We analyzed the iTRAQ samples with a LTQ Orbitrap using “higher energy collision‐induced dissociation” fragmentation [Olsen, J. V., et al. , Nat. Methods 2007, 4 , 709–712] and quantified with Proteome Discoverer v.1.1 (Thermo Fisher Scientific). We believe that the presented protein mixtures will be useful to assess the performance of the iTRAQ‐based quantitation proteomic strategy in any laboratory.