Protein Quantitation

Protein Quantitation

The Proteomics and Metabolomics Shared Resource offers relative protein quantitation based on several 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 quadrupole time-of-flight (Q-ToF) 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 including 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 liquid chromatography (LC) prior to tandem mass spectrometry (MS/MS).

Open, unbiased ('omic) differential protein expression can also be performed using isotope labeling technology for parallel processing of up to four samples (iTRAQ 4-plex) within a single LC-MS/MS or LC/LC-MS/MS experiment. Data dependent acquisition on a Q-ToF 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 a 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 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 Q-ToFs.