msb-quantworks

iTRAQ/TMT

Chemical labeling methods such as iTRAQ and TMT enable increased throughput in quantitative proteomics experiments. Samples are digested with trypsin and are subsequently labeled with isobaric tagging reagents, tagged samples are then pooled prior to analysis. The tag chemistry is such that tagged peptides from different samples in a pool will have the same precursor ion m/z and under collision induced dissociation conditions will yield diagnostic tag specific reporter ions that can be used for quantitation. Thus a peptide fragmentation spectrum contains not only information on the identity of the peptide but also information on the relative amounts of that peptide in each pooled sample. Data processing software is used to interpret the peptide level data and report quantitative results for detected proteins.

To improve the sensitivity of the chemical labeling techniques we use off-line strong cation exchange (SCX) chromatography to fractionate the pooled sample prior to LC/MS/MS analysis.

The steps involved in a chemical labeling experiment are:

  • Sample preparation – cell or tissue lysis, plasma or serum depletion, urine and CSF sample clean-up; protein quantitation on all samples.
  • Sample digestion – reduction and alkylation followed by in-solution trypsin digestion.
  • Sample labeling – Reagents are available that allow the multiplexing of 2, 4, 6 or 8 samples.
  • Sample pooling – equal amounts of labeled samples are combined.
  • Fractionation –fractionation of pooled sample using off-line SCX.
  • Nano LC/MS/MS – 250 nL/min liquid chromatography to yield high sensitivity; interfaced to a state of the art Velos Orbitrap Pro tandem mass spectrometer.
  • Data analysis – database searching with Mascot, data validation, visualization and quantitation using Scaffold Q+.
  • Report generation – PDF report and Excel. Data rationalized and presented to ensure clarity and the ability to understand the results.

Key features of a chemical labeling experiment are:

  • High throughput.
  • Applicable to any biological system.
  • Multiplexed for 2, 4, 6 or 8 samples in a single experiment

For more information on Thermo Scientific Pierce chemical labeling reagents visit www.thermoscientific.com/pierce