Protein sequencing stable isotope labeling

Enzymatic digestion of proteins in 1 1 0/ 0 water has been applied to facilitate de novo sequencing [136]. The labelling results in split peaks with a 2 Da difference for the y-ions, enabling easy discrimination between peaks due to b- and y-ions. Other stable isotope labelling methods (Ch. 18.4.2) involving modification of either the N- or the C-terminal can assist in de novo sequencing as well. 

For successful NMR experiments a protein solution of high purity (>95%), stability (over a week), and appropriate concentration (0.1-1 mM) is needed the total sample volume and mass should be 350-550 uL and 3-30 mg, respectively. 2,2-Dimethyl-2-silapentane-5-sulfonic acid (DSS) rather than tetramethylsUane (TMS) is used as a reference compound. Although both natural and synthetic polypeptides and proteins can be used, most samples are expressed via a suitable prokaryotic or eukaryotic cellular or ceU-free recombinant technique. The latter method eliminates most toxic effects attributed to the overproduction of recombinant proteins. Biotechnological approaches have the common advantage of large-scale protein production, specific or non-specific stable isotope labeling, and easy sequence variation. 

The above facts do not favour protein or peptide quantitation using MALDI-MS. Some problems are associated with MALDI-MS quantification i) low shot-to-shot reproducibility, ii) various signal suppression effects, and iii) strong influence of sample preparation and matrix crystallisation. Nevertheless, it is possible to use MALDI-MS to obtain absolute or relative quantitation. In most cases, the idea is to use an internal standard for an absolute quantitation, but this standard must have the same physico-chemical characteristics as the quantified peptide. The use of a different peptide in terms of sequence may result in different desorption and ionisation properties. Usually, the internal standard is the same peptide labelled with a stable isotope to modify slightly the mass. 

---