Proteomics

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.

1. analyze separate samples and then compare separate sets of data
2. label different samples, and analyze them together
3. analyze samples separately with peptide standards

Method 1 is routine use because of advances in bioinformatics. It requires reproducible separations of peptides

Method 2 requires more up front work but can have more accurate results.

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.

1. growing cells so they incorporate an isotope normally in low abundance e.g. N15, C13
2. chemical labeling with TMT labels

Method 3, using TMT is being used here. 6-18 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.