Development of novel guanidino-labelling derivatisation (GLaD) reagents for liquid chromatography/matrix-assisted...

A new generation of guanidino-labelling (GLaD) reagents were developed for quantitative proteomics using offline microcapillary liquid chromatography (LC) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In order to reduce the unwanted overlapping between the isotopic envelopes of the two differentially labelled peptide ions, a novel synthetic route was described for production of both (13)C- and (15)N-containing isotopomers of N,O-dimethylisourea. The use of these types of isotopes has no deleterious effect on the retention times of both differentially labelled peptides during offline microbore reversed-phase LC. In addition, the possibility to incorporate a mass difference of 4 Da can be exploited during post-source decay analysis to generate product ion spectra in which fragment ions containing the modifications appear as doublets in the corresponding product ion spectra, thus facilitating identification of the C-terminal fragment ions.