Enhanced Diagnostic Tools
Isotopes refer to differences in the number of neutrons in the nucleus of atoms; isotopes can be radioactive, when they decay, releasing energy in the process and stable isotopes, as the name indicates, do not decay and actually can be isolated as such. Most biologically active proteins have two or more stable isotopes, with the lightest ones representing the most abundant ones. For example C13 and N15 are heavy isotopes with an abundance of 1% and C12 and N14 have an abundance of 99%.. In the case of carbon, there is also a radioactive form C14.Stable isotopes can be introduced in to amino acids, by feeding growing bacteria with the corresponding precursor, containing the desired element. In turn these amino acids can be used to prepare the corresponding Fmoc protected amino acids, that then can be incorporated during peptide synthesis into specific positions of the given peptide/protein.
MALDI-TOF of small peptides may not easily detect the difference in mass, but in larger proteins one can more readily detect the differential mass distribution due to the presence of the heavy isotope. The biological researcher can introduce at will stable isotopes into the sequence of their interest, and this is very helpful in studying protein-protein interactions,using methods, such us proton NMR as an example. One challenge in using heavy/stable isotopes is their high cost, due to the cost involved during the enrichment process. Most masses given for peptides correspond to the calculated monoisotopic masses. For refined and/or special applications, the biological researcher will need to take into account the heavy isotope masses, and as we mention before, the heavier the protein the more noticeable the increased differential in the molecular weight of the protein in question. Synthetic peptide chemistry has proven very valuable as it allows the production of milligram to gram quantities of stable isotope containing peptides, where selected amino acids can at will contain the corresponding stable isotopes of carbon, hydrogen or nitrogen.
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