Heavy Isotope Labelling

Deuterium labelling is the most useful for mass spectrometry. A single atom incorporated increases the intensity of the M+1 ion. There must be sufficient present to make the ion intensity greater than that due to the natural abundance of C (1.1%) and any other isotopes. Natural (0.015%), N (0.36%) and O (0.04%) add little to the strength of the M-l-1 ion, and 0 (0.2%) adds little to the M+2 ion. Incorporation of an intact group will produce a new ion at M+3 that can be readily seen and its intensity measured to give the degree of incorporation. 

In Chapter 3 an example was given of the demonstration that isobutyric acid (and methacrylic acid) in an insect was derived from valine by 

Alternatively, the experiment can be followed by C NMR spectroscopy. We can observe coupling in the proton-decoupled 

---

Labeling with 2H, 13C, 15N and/or lsO can be introduced via peptide synthesis, cell culture, or hydrolysis in labeled water [88]. The heavy isotope-labeled peptide can be used as an IS to obtain quantitative measurements of the protein concentration. Typically, the protein sample of interest is digested with trypsin, and the isotope-labeled control peptides are added to the mixture. The signature peptides in the digest can be separated and quantified by HPLC-ESI-MS/MS. Alternatively, MALDI-MS can also be used for tryptic peptide determinations after some separation steps such as gel electrophoresis [89]. 

The value of heavy isotope labelling is illustrated in an investigation of the metabolite pattern of (+)- propoxyphene in man [394]. Three different deuterium labelled forms of propoxyphene were prepared, dv (25), d3 (N-CD3), and di (N-CHD2). In the initial screen, a 1 1 mixture of undeuterated propoxyphene (do) and its dy analogue was administered. 

The aldehyde group of aldoses can either be oxidized or reduced and ketoses can be reduced. The best laboratory reagent for reduction is sodium boro-hydride which acts rapidly in neutral aqueous solutions (see Eq. 4-2). Since both NaB H4 and NaB H4 are available, radioactive or heavy isotope labels can be introduced in this way. The aldehyde groups can be oxidized by a variety of agents to the corresponding aldonic acids, a fact that accounts for the reducing properties of these sugars. In alkaline solution aldoses reduce Cu + ions to cuprous oxide (Eq. 4-3), silver ions... 

Isotope Labeling. Heavy isotope labels, alone, would very likely not produce better results than radioisotope labels. Problems of isolation and purification would be more difficult than radioisotope studies due to the nature of the detection system which would be needed, mass spectrometry. However, the combined use of heavy isotope and radioisotope labeling can provide very definitive information on confirming the identification of suspected metabolites in the same manner as using dual radioisotopes, such as and H, or and How-... 

Little fragmentation occurs during NICI, and this mode of ionization is generally employed for quantitative analyses of trace amounts of compounds of known structure in conjunction with the use of heavy isotope-labeled internal standards. 

---