Controlling deuterium isotope effects in comparative proteomics.

This paper focuses on identifying structural features responsible for resolution of heavy isotope coded peptides during reversed-phase chromatography. This was achieved by using labeled coding agents that varied in structure, number of deuterium atoms, placement of deuterium in the coding agent, and the functional group targeted by the reagent. Six coding agents were examined. Deuterated versions of the coding agents studied included succinic anhydride-2H4, acetic acid 2,5-dioxopyrrolidin-1-yl ester-2H3, propionic acid 2,5-dioxopyrrolidin-1-yl ester-2H5, pentanoic acid 2,5-dioxopyrrolidin-1-yl ester-2H9, [3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylammonium chloride-2H9, and the commercial ICAT-2H8 reagent. It was found that these labeling agents vary widely in both their absolute and relative contribution to the chromatographic isotope effect. Relative effects were evaluated by normalizing resolution for the number of deuterium atoms in the derivatized peptide. The single, most dominant effect was the placement of deuterium atoms relative to hydrophilic functional groups in the coding agent. It was concluded that the probability of a deuterium atom interacting with the stationary phase of a reversed-phase chromatography (RPC) column and impacting resolution is greatly diminished by placing it adjacent to a hydrophilic group, as explained by solvophobic theory. But peptide size and coding agent size were also seen to correlate inversely with the magnitude of the isotope effect. This effect was explained as being due to the relative size of the coding agent versus that of the coding agent-peptide conjugate.