Quantitative Proteomics - Relative and Absolute Quantitative Proteomics

Relative and Absolute Quantitative Proteomics

Mass spectrometry is not inherently quantitative because of differences in the ionization efficiency and/or detectability of the many peptides in a given sample, which has sparked the development of methods to determine relative and absolute abundance of proteins in samples. The intensity of a peak in a mass spectrum is not a good indicator of the amount of the analyte in the sample, although differences in peak intensity of the same analyte between multiple samples accurately reflect relative differences in its abundance. One approach for relative quantitation is to separately analyze samples by MS and compare the spectra to determine peptide abundance in one sample relative to another, as in label-free quantitation strategies. An approach for relative quantitation that is more costly and time-consuming, though less sensitive to experimental bias than label-free quantitation, entails labeling the samples with stable isotope labels that allow the mass spectrometer to distinguish between identical proteins in separate samples. One type of label, isotopic tags, consist of stable isotopes incorporated into protein crosslinkers that causes a known mass shift of the labeled protein or peptide in the mass spectrum. Differentially labeled samples are combined and analyzed together, and the differences in the peak intensities of the isotope pairs accurately reflect difference in the abundance of the corresponding proteins. Absolute proteomic quantitation using isotopic peptides entails spiking known concentrations of synthetic, heavy isotopologues of target peptides into an experimental sample and then performing LC-MS/MS. As with relative quantitation using isotopic labels, peptides of equal chemistry co-elute and are analyzed by MS simultaneously. Unlike relative quantitation, though, the abundance of the target peptide in the experimental sample is compared to that of the heavy peptide and back-calculated to the initial concentration of the standard using a pre-determined standard curve to yield the absolute quantitation of the target peptide.

Relative quantitation methods include:

• Isotope-coded affinity tags (ICAT)
• Isobaric labeling
  • Tandem mass tags (TMT)
  • Isobaric tags for relative and absolute quantitation (iTRAQ)
• Label-free quantification
• Metal-coded tags (MeCATs)
• N-terminal labelling
• Stable isotope labeling with amino acids in cell culture (SILAC)

Absolute quantitation is performed using:

• Selected Reaction Monitoring (SRM)

MeCAT can be used in combination 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.