MS-based Metabolomics and Proteomics
Even though the MS-based analysis of metabolites and proteins relies on the same principles (basically the measurement of an exact mass), these two disciplines are quite different in analytical demands. This diversity is based on the fact that proteins (or better peptides), which are polymers of 20 amino acids, are less chemically diverse between each other than the highly heterogeneous metabolites. Therefore the major challenge in proteomics requires overcoming the problem of enriching and measuring the rare and hard to be extracted proteins (e.g. transcription factors and membrane proteins), while the problems of metabolite analysis is more complex.
A reliable peak annotation and the discrimination of biological from non-biological compounds is the main limitation of MS-based metabolomics. We can overcome this problem by analyzing three differentially isotope-labeled metabolomes (13C, 15N and 34S ) and comparing it to an unlabeled sample.
As a consequence, the analysis of metabolites requires not only a larger repertoire of extraction and separation methods, also due to the fact that metabolomics benefit only indirectly from completed genome sequencing projects the identification of the measured compounds is still the limiting factor. The reason for this discrepancy is that RNA and proteins are essentially collinear with genomic information, while metabolites represent a completely different level of genome information realization which does no longer display collinearity to the genome sequence.
Therefore the main limitation of MS-based metabolomics lies still in the reliable peak annotation and the discrimination of biological from non-biological compounds. The best way to reliably annotate a measured compound is to cross check it with an authentic standard. However, in many cases, especially for complex plant secondary metabolites, reference compounds are not always available. To circumvent this problem we have made use of an isotope-labeling-based metabolomic analysis strategy where three differentially isotope-labeled metabolomes (13C, 15N and 34S) are analyzed in parallel to an unlabeled sample. This strategy, which is further explained in the subsection isotope labeling, allows to measure the same compound with specific mass shifts, enabling a reliable elemental composition annotation.