Proteomics has been defined as the qualitative and quantitative comparison of proteomes under different conditions to further unravel biological processes
Mass spectrometry can compare samples in one or a few complex analyses, giving more information for less human effort than 2-D gels. We have three ways of comparing samples.
- analyze separate samples and then compare separate sets of data
- label different samples, and analyze them together
- analyze samples separately with peptide standards
Method 1 is getting closer to routine use because of advances in bioinformatics. It requires reproducible separations of peptides
Method 2 has been in use for longer.
Methods 1 and 2 are for discovery of proteins which differ between two or more samples.
Method 3 is is for situations to measure the amounts of specific, known proteins in different samples. It can give absolute quantitation of specific proteins and requires preparing isotopically labeled peptides. This method of Selective Reaction Monitoring/Multiple Reaction Monitoring is described separately.
Methods for labeling different samples
- growing cells so they incorporate an isotope normally in low abundance e.g. N15
- reactions to exchange deuterium for hydrogen
- chemical labeling with TMT, iTRAQ or ICAT reagents
Method 3, using iTRAQ is being used here. Up to 6 different samples can be analyzed at one time.
The principle is to digest a protein mixture to peptides. Then label peptides on the N-terminal with one of the similar reagents. Combine the samples for further preparation and analysis.
In the mass spectrometer, the label fragments. The low mass part of the label differs from the other labels; relative amounts of the low mass labels are used to quantitate the peptides, and hence the proteins they came from.
Two dimensional gels are a traditional method to compare proteins from different tissues. The technology is easily understood, there are comprehensible images to look at, the equipment is not greatly expensive.
However, getting reliable data requires a skilled operator who may spend months developing the skill, the dynamic range of the technique is limited, it has biases against large and basic proteins, and a successful analysis may require dozens of individual protein identifications by mass spectrometry.