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Relative Quantification: iTRAQ™

iTRAQ™ is a multiplexed protein quantitation strategy that provides relative measurements of proteins in complex mixtures. For quantitative protein analysis the isobaric mass labels are covalently attached at the N-termini and lysine side chains of peptides in a digest mixture. The reagents are differentially isotopically labeled such that all derivatized peptides are isobaric and chromatographically indistinguishable, but yield signature or reporter ions following CID that can be used to identify and quantify individual members of the multiplex set. The iTRAQ experiments can be done in 2, 4, 6, or 8 plex set up. In an 8 plex you can have 8 unique samples.

iTRAQ

Relative Quantification: Tandem Mass Tag™ (TMT) Systems

The Thermo Scientific™ Tandem Mass Tag™ Reagents are designed to enable identification and quantitation of proteins in different samples using tandem mass spectrometry (MS). The TMT10plex Label Reagents share an identical structure with TMTzero, TMTduplex, and TMTsixplex Reagents but contain different numbers and combinations of 13C and 15N isotopes in the mass reporter.

  • Powerful—concurrent MS analysis of multiple samples increases sample throughput and enables relative quantitation of up to 10 different samples derived from cells, tissues or biological fluids
  • Consistent—identical reagent structure and performance among TMTzero, TMTduplex, TMTsixplex and TMT10plex Reagents allow efficient transition from method development to multiplex quantitation
  • Robust—increased multiplex capability results in fewer missing quantitative values
  • Flexible—multiple chemistries allow functional group specific labeling and enrichment, using anti-TMT immobilized resin
  • Convenient—three fill sizes (0.2, 0.8, and 5 mg) to accommodate small volume, high throughput to bulk labeling needs
  • Efficient—amine-, cysteine- and carbonyl-reactive chemistries utilize an effective strategy for efficient labeling of target functional group
  • Compatible—optimized for use with high-resolution MS/MS platforms using SP2 or SPS MS3 quantitation mode with data analysis fully supported by Thermo Scientific™ Proteome Discoverer™ 2.1 Software
  • Procedure summary for MS experiments with Thermo Scientific™ TMT10 or TMT16 or TMT18plex Isobaric Mass Tagging Reagents. Protein extracts isolated from cells or tissues are reduced, alkylated and digested overnight. Samples are labeled with the TMT Reagents and then mixed before sample high pH reverse phase fractionation and clean up. The results are 8 unique peptide fractions  that are each analyzed by high-resolution Orbitrap FAIMS LC-MS/MS (30 cm C18 1.7 micron particle size column and 120 min nano-UPLC gradients (Vanquish Neo)). The instrument files are then pooled for analysis to identify peptides and quantify reporter ion relative abundance and produce quantifications tables using PEAKS analysis software.