Quantitative proteomics
Overview
The aim of quantitative proteomics is to obtain quantitative information about all proteins in a sample. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about differences between samples. For example, this approach can be used to compare samples from healthy and diseased patients. The methods for protein identification are identical to those used in general (i.e. qualitative) proteomics, but include quantification as an additional dimension.
Mass spectrometry-based proteomics
Due to differences in ionization efficiency and/or detectability the intensity of a peak in a mass spectrum is not a good indicator of the amount of the analyte. However, differences in peak intensity of the same analyte accurately reflect differences in its abundance. Therefore, in mass spectrometry-based proteomics relative quantification is used to compare protein abundance between samples. This can be achieved by labeling one sample with stable isotopes which leads to a mass shift in the mass spectrum. Differentially labeled samples are be combined and analyzed together. The differences in the peak intensities of the isotope pairs accurately reflect difference in the abundance of the corresponding proteins. Current methods include stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tags (ICATs) and metal coded tags (MeCATs). MeCAT can be used in combinantion with element mass spectrometry ICP-MS allowing first time absolute quantification of the metal bound by MeCAT reagent to a protein or biomolecule. Thus it is possible to determine the absolute amount of protein down to attomol range using external calibration by metal standard solution. It is compatible to protein separation by 2D electrophoresis and chromatography in multiplex experiments. Protein identification and relative quantification can be performed by MALDI-MS/MS and ESI-MS/MS.
Two-dimensional gel electrophoresis
Modern day gel electrophoresis research often leverages software-based image analysis tools primarily to analyze bio-markers by quantifying individual, as well as showing the separation between one or more protein "spots" on a scanned image of a 2-DE product. Differential staining of gels with fluorescent dyes (difference gel electrophoresis) can also be used to highlight differences in the spot pattern.
References
- Ong SE, Mann M (2005). "Mass spectrometry-based proteomics turns quantitative". Nature Chemical Biology. 1: 252–262. PMID 16408053.