Beschreibung
To deal with the challenges in quantitative proteomics, Metal-coded affinity tagging (MeCAT) was developed for absolute and high sensitive quantification of proteins and peptides. It labels biopolymers with lanthanide ions that are loaded into 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) complexes instead of isotopes. Different MeCATs have been developed and successfully applied to quantify the peptides and proteins. However, due to the large size of the DOTA complexes and spatial hindrance in biopolymers, some active sites are inaccessible, which limits its wide application. In this book, a new MeCAT label based on in situ Click Chemistry (MeCAT-Click) is present for improving the labeling efficiency and reducing the steric hindrance. The proposed label showed compatibility of ESI-MS techniques allowing clear identification of the labeled peptides and proteins. Different approaches for analysis of the MeCAT-Click labeled samples have been applied such as, liquid chromatography separations or gel electrophoresis followed by mineralization and direct infusion ICP-MS, both with intact and digested proteins. In addition to the application to standard samples, it was further utilized to quantify the proteins during the heat shock in Escherichia coli, which shows its capability of quantifying the proteins in real samples. During the application, two parallel experimental approach, where both elemental and molecular MS techniques were involved in a complementary way. Moreover, the quantification of peptides labeled with MeCAT-Click was proved using characteristic fragments in ESI-MS/MS, whose results agreed with those obtained by ICP-MS and from the full scan spectra of the parent ions by ESI-MS. Finally, the concept of peptide quantification using the elemental reporter ions was also proved.
