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iTRAQ and TMT

These techniques use multiplexed isobaric mass tags to label peptide digest mixtures. iTRAQ (AB SCIEX) has 4 or 8 complementary reagents consisting of an amine reactive group (binds to N-terminal and the epsilon amino group of Lysine), a reporter group (with masses of 114-117 for the 4 plex and 113-119; and 121 for the 8 plex), and a “balance” group (keeps the tag mass at a constant 145 Da for the 4 plex and 304 Da for the 8 plex). The TMT tag (Pierce) is similar to iTRAQ but with amine, cysteine or carbonyl reactive tags, a mass reporter region (6 different tags with masses 126 to 131) and a mass “normalizer” region. For the amine reactive tag, the normalizer region keeps the tag at a constant mass of 229. Hence, using these tags, 2- 8 plex multiplexed experiments can be performed. The tags are “silent” in the MS mode, with quantitation performed on the reporter ions in the MS/MS spectra. In order to delve deeper into the proteome, these protein profiling experiments can be combined with 1) SCX fractionation, as in a MudPIT experiment ;or a C18 reverse phase fractionation step, or 2) run in duplicate. On average, fractionation can increase the proteome coverage >50% and the duplicate run, more than 20% (an additional fee is associated with the duplicate run).

Grouped Service

Suitable Protein Complexity

Analysis Includes

iTRAQ-4 plex Compares/quantifies 4 samples Sample clean-up step using an extraction or precipitation technique, a dual enzymatic digestion, amino acid analysis protein quantitation on each sample, chemical labeling, a long LC-MS/MS gradient run, a database search and data analysis. Grouped costs with fractions also include a strong cation fractionation or C18 reverse phase separation into 10 fractions which are run individually on a long length gradient.
iTRAQ-4-plex-fractions (MudPIT) adds in fractionation into 10 fractions for separate LC-MS/MS analysis
TMT-6-plex Compares/quantifies 6 samples
TMT-6-plex-fractions (MudPIT) adds in fractionation into 10 fractions for separate LC-MS/MS analysis
iTRAQ-8-plex Compares/quantifies 8 samples
iTRAQ-8-plex-fractions (MudPIT) adds in fractionation into 10 fractions for separate LC-MS/MS analysis

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