Quantitation of changes in protein abundance is key to discovering novel biomarkers. Currently, reverse phase liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) can be used to quantify changes in protein expression levels. 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. In this context, we describe two different samples (4-protmix and 8-protmix) suitable for relative protein quantitation using isobaric Tags for Relative and Absolute Quantitation (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 10-fold differences in relative abundance.The two reference samples proposed here (4-protmix and 8-protmix) cover a wide range of protein fold changes and protein concentrations, respectively, which can be used to optimize the settings used during acquisition with different mass spectrometers and to test the performance of different quantitation algorithms used for iTRAQ experiments.Three technical replicates corresponding to 800 fmol of 4-protmix and 8-protmix were analyzed by HPLC-MS/MS using two platforms, a ChipLC coupled to a 6530 Q-ToF (Agilent Technologies) and a 1200 nano-HPLC (Agilent Technologies) coupled to -sult, the analysis of the 4-protmix and the 8-protmix all proteins in the samples (Tables 1 and 2 As shown here, we were able to detect up to 24-fold changes in protein ratios. The standard de--sponded to the highest protein fold change in our samples (24-fold).
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