Quantification of proteins
Mass spectrometry in itself is a quantitative tool allowing the quantification of peptides in parallel with their identification. Relative quantification is now widely used to identify changes in protein levels or levels in post-translational modifications. The most basic approach is called spectral counting. More accurate values are achieved by peptide or fragment peak integration. Best results are obtained with isotopic labeling. However, if larger changes in protein levels are expected, label-free approaches are cheap and attractive alternatives.
We have established an automated data analysis pipeline based on MASCOT and Scaffold (spectral count) and complemented it with
- ProgenesisLC for non-labeled quantitative proteomics approaches
- Proteome Discoverer for Heavy Peptide techniques (SILAC, TMT, iTRAQ)v
Multiple reaction monitoring (MRM) is another, targeted method for isotopic or non-labeled peptide quantification. We have developed an LC-MRM method for absolute quantification of histone H3 peptides, which differ in their state of methylation and acetylation. In addition, LC-MRM can be used to quantify relative levels of other posttranslational modification like phoshorylation or glycosylation.