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. Author manuscript; available in PMC: 2016 Jan 4.
Published in final edited form as: Methods Mol Biol. 2011;756:99–130. doi: 10.1007/978-1-61779-160-4_5

Fig. 2.

Fig. 2

Principle of isobaric mass-tags in quantitative mass spectrometry. (a) Several combinations of different-sized reporters of iTRAQ tags facilitate quantification of up to 8 different samples (masses from 113 to 121, excluding 120 as this corresponds to phenylalanine). Quantitative information is obtained from relative intensities of reporter ions in MS/MS spectrum. TMT (tandem mass tag: Thermo Electron Corporation) has the same property with iTRAQ but has different reporter and balancer chemistry. (b) In SILAC, isobaric amino acids are metabolically incorporated into all the cellular proteins. Animals can be fed and bred through multiple generations using feed with differential amino acid composition [SILAM: 60]. The equal amount of samples are combined and then applied to LC-MS/MS analysis. Quantitative information is obtained from relative intensities of light- and heavy-peptide ions in MS spectrum. (c) A representative analytical procedure of quantitative MS. In the bottom-up approach, complex peptide mixtures are fractionated through strong cation-exchange chromatography (SCX), which is essential for reducing sample complexity and increasing the number of identified peptides. Each fraction is analyzed through reverse-phase (RP) LC-MS/MS. For the nonisotopic study, quantitative information is obtained through peak intensity of specific peptides in ion chromatogram and more widely through counting finally matched MS/MS spectra and statistical manipulation. In case of using isobaric-tags, differentially labeled samples are combined before SCX chromatography. Quantitative information is obtained from MS or MS/MS spectrum, dependent on the property of isobaric tag. (d) Modes of sample preparation, labeling, and mixing for MS analysis. For mass-tag labeling procedures such as iTRAQ the individual extraction of proteins, then peptides from each sample is followed by individual mass-tag labeling and then mixing for single-run MS analysis. For stable isotope incorporation procedures, sufficient cell passages or animal generations in the presence of differential isotopes is required before mixing for single-run MS analysis.