Using the ability to multiplex samples with Tandem Mass Tags®, TMTcalibrator™ uniquely combines fluids and tissues or cells lines to bias biomarker discovery towards disease-related biomarkers. The use of an excess of disease tissue sample ensures that all representative biomarkers are present in each analysis and forces the mass spectrometer to mostly select tissue-derived peptides for analysis. Because the same peptide present in the body fluid will also be selected for analysis at the same time, due to the isobaric TMT® labelling, we substantially increase sensitivity and reduce the frequency of peptides from the normal higher abundance fluid proteins.
In biomarker discovery studies TMTcalibrator™ requires two types of sample;
In validation studies, synthetic or recombinant peptides can also be used as the calibrant sample.
Yes, synthetic or recombinant peptides can be used when using TMTcalibrator™ for validation studies or as an absolute qualification method.
TMTcalibrator™ usually requires around 50 – 100 micrograms of protein from each fluid sample and around 800 micrograms of protein for the calibration sample. If combining TMTcalibrator™ with phosphopeptide enrichment we require approximately 10 times this amount for both fluids and the calibrant.
For TMTcalibrator™ we typically use LC-MS/MS (MS2) running on UHPLC-Orbitrap Fusion Tribrid, ThermoScientific.
Yes. We have successfully combined TMTcalibrator™ with metal affinity enrichment of phosphopeptides. In principle any enrichment method can be followed, though careful consideration must be given when using immunoprecipitation methods. Please contact us discuss your specific requirements.
It is possible to add an include list to any of our mass spectrometry methods however for TMTcalibrator™ an include list would not normally be required. This is because the calibrator trigger provides excellent protein coverage without the need to run include list that can reduce the total number of peptides & proteins identified.
TMTcalibrator™ typical performance stats;
The peptides & proteins that are identified in TMTcalibrator originate from three groups;
It is the proteins within this final group that are the best candidate fluid biomarkers of disease.
Sensitivity of TMTcalibrator™ will vary from peptide to peptide and is influenced by the efficiency of trypsin digestion of the parent protein and the ease of ionisation of the TMT®-labelled peptide relative to other peptides eluting at the same time. We have been able to detect peptides from proteins known to be at concentrations of around 100 pg/ml in CSF and similar low abundance targets in plasma such as myelin basic protein, neurogranin and PDGF receptor alpha.
The standard data output of TMTcalibrator™ is QuantSheet™, a Microsoft Excel file. We can also provide other formats such as .CSV or .txt on request. Please discuss specific requirements with our sales team.