Isotope-Coded Derivatization (ICD)

What is ICD Technology

Isotope-coded derivatization (ICD) is a promising alternative to intraisotopic technology, that can be used to overcome matrix effects caused by coexisting substances that are common in liquid chromatography-mass spectrometry/mass spectrometry analysis of generations. The stable isotope-coded derivatization (ICD) technique utilizing mass-different isotope tags is known to be an efficient means for metabolite profiling study. The purpose of the ICD method is to obtain the reliable quantitative data of multiple components in complex matrices. This method is efficient for the differential analyses in proteomics and metabolomics. The outline of the method is shown in Fig. 1.

Fig. 1 Schematic flow diagram of ICD method

Disadvantages of ICD Method and Improvement Methods

A pair of stable isotope reagents, such as light and heavy reagents labeled with H/D or ¹²C/¹³C, is required for the ICD method. Although the ICD method is excellent means for the differential analysis, the disadvantages such as isotope effect are lying on the ICD. It is well known that the stable isotopes into peptides affect their retention time under reversedphase chromatographic condition. The isotope effect of H/D is currently inevitable. However, the effect is possible to reduce by decrease the number of D in the ICD structure. Furthermore, a deuterium atom interacting with the stationary phase chromatography column is diminished by placing it adjacent to a hydrophilic group, as explained by solvophobic theory. In contrast, the chromatographic behavior of ¹²C/¹³C labeled derivatives is almost comparable and the isotope effect is negligible. Same no resolution of peptide isoforms has been observed during reversed-phase chromatography when ¹⁵N was used as coding agent. In spite of the drawback, the ICD method is attractive means in not only proteomics but also metabolomics. Although the ICD method has been applied to the proteomics, the technique is also excellent means for metabolite profiling study.

The Following Features Are Required For Ideal ICD Reagents:

1. The ICD reagents including "light" and "heavy" isotopes can be commercially available or easily synthesized.
2. The derivatization reaction is simple under mild conditions and no side reaction occurs.
3. Both light and heavy reagents show not only the same reaction time course, but also the same reaction yields.
4. The ICD is equally reacted with the compounds possessing the same functional group to produce the corresponding derivatives.
5. The resulting derivatives can be separated by reversed-phase chromatography, even for polar or ionic molecules.
6. The derivatives can be efficiently ionized and detected by electrospray ionization mass-spectrometry (ESI-MS).
7. The effective large shift in the detection mass is provided for avoiding any signal interference raised from low mass background substances and intrinsic contaminants commonly present in the ESI process.
8. The analysis tagged with the light and heavy reagents do not show drastic isotope effect in the chromatographic separation and eluted in the same position.
9. The same daughter ion derived from the ICD reagent appears on the MS spectra independent of the structure of the parent analysis. The typical and strong m/z signal corresponding to the ICD reagent itself and/or a part of the ICD reagent is preferable for the selected reaction monitoring (SRM) analysis, because the simultaneous determination of a series of molecules having the same functional group in a sample is possible by precursor ion scanning.