Protein Quantitation

The Proteomics Facility Core offers a range of methods for protein quantitation.

Triple quadrupoles are built with three quadrupole mass spectrometers arranged in tandem. By careful tuning, one can determine which ions are capable of transversing the instrument. For selected reaction monitoring (SRM), the first quad (Q1) is tuned to select parent ion(s) of interest. Those ions pass into Q2, a collision chamber which induces peptide fragmentation. The fragment ions then pass into Q3, which monitors for the presence of ions characteristic of the parent molecule (called 'transitions'). A key advantage of SRM/MRM is that, unlike many mass spec approaches, this technique is quantitative, not qualitative. A given ion's spectral signal can be converted into abundance by use of a standard curve and/or internal standards. In practice, one to hundreds of transitions can be monitored in a single chromatographic run.

Another label-free method of quantitation is spectral counting, which compares the number of spectra identified for a given peptide in different biological samples.

iTRAQ (Applied Biosystems) or TMT (Thermo Scientific) reagents provide an alternative approach to protein quantitation.  In this method, samples are digested and differentially labeled with isobaric tags. Unique reporter ions can then be relatively quantitated within the MS2 fragmentation pattern of labeled peptides.

For SILAC (stable isotope labeling with amino acids in cell culture), two populations of cells are cultivated, except one is fed normal growth medium, and the other with medium containing amino acids labeled with stable (non-radioactive) heavy isotopes. Pairs of chemically identical peptides of different isotopic composition can then be differentiated in the mass spectrometer owing to their mass difference.