The Proteomics Facility offeres relative protein quantitation based on several different paradigms, including label-free expression analysis, isotope labeling expression analysis (iCAT or iTRAQ), and targeted protein expression.
Open, unbiased ('omic) differential protein expression can be performed using label-free technology with MSE acquisition and IdentityE data processing, on any of our nanoacquity UPLC systems coupled to QTof mass spectrometers. For these experiments we recommend triplicate analysis of each biological replicate, to increase statistical significance of the peptide (and thus protein) fold-changes measured. This type of experiment can be extended to studies include protein expression as a function of time given an external stimuli or gene knockout, for instance. Label-free differential expression is only available on separations utilizing a single dimension of LC prior to MS/MS.
Open, unbiased ('omic) differential protein expression can also be performed using isotope labeling technology for parallel processing of up to 4 samples (iTRAQ 4-plex) within a single LC-MS/MS or LC/LC-MS/MS experiment. Data dependent acquisition on a QTof mass spectrometer and MASCOT database searching is used with isotope-labeling experiments. Differential expression utilizing isotope labeling can be performed with either single dimension (LC-MS/MS) or multidimensional (LC/LC-MS/MS) separations.
For targeted protein expression experiments, a triple quadrupole mass spectrometer operating in multiple-reaction-monitoring (MRM) mode is used to detect very specific signals from the target peptides of interest. This technique is approximately one order of magnitude more sensitive than the open ('omic) approach and provides improved quantitative precision. However, only the targeted peptides are quantified. MRM mass spectrometry is the best way to verify fold-change data obtained from open ('omic) differential expression studies on the QTofs.