Fragmentation behavior of metal‐coded affinity tag (MeCAT)‐labeled peptides

Abstract

Quantitative proteomics has become an important method in modern life sciences. Besides protein identification, the aspect of quantification is of rapidly increasing relevance. MeCAT (metal-coded affinity tagging) is able to provide a tool that enables relative as well as absolute quantification. For structural elucidation, knowledge on the fragmentation behavior of MeCAT-modified peptides is highly beneficial. Therefore, the fragmentation behavior of MeCAT-labeled peptides under collision induced dissociation (CID), electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) conditions was studied. Application of CID and ECD allowed a straight-forward sequence elucidation of MeCAT-labeled peptides. During IRMPD all MeCAT-labeled peptides form characteristic fragments resulting from the fragmentational cleavage of the tagging group, thus, providing a screening method for the identification of labeled compounds. Furthermore, occurring side reactions during the labeling process were investigated. By-products were structurally characterized and reaction conditions were optimized in order to prevent the formation of these.

Elemental labelling combined with liquid chromatography inductively coupled plasma mass spectrometry for quantification of biomolecules: A review

“…However, subsequent work investigated analytical robustness and suitability of this concept for biological applications [119]. Additionally, Pieper et al [120] from the same group examined the fragmentation behaviour of MeCAT labelled BSA utilizing nano or capillary LC–MS for detection. Moreover, a iodoacetamide reactive group combined with the MeCAT lanthanide label was employed for quantification of digested BSA and β-lactalbumin via SDS-PAGE or nano-RP-ESI-MS/MS (applying a Zorbax 300 SB C18 column) revealing higher labelling efficiency compared to the maleimide functionalized MeCAT approach.…”

MeCAT peptide labeling for the absolute quantification of proteins by 2D‐LC‐ICP‐MS

“…The application of the MeCAT labeling strategy to standards and real samples has already been published . The robustness of the analytical methodology, the stability of the label and its suitability for biological applications have been demonstrated, as well as the analytically useful fragmentation behavior of the labeled peptides . Recently, the application of MeCAT labeling and ICP‐MS detection for the absolute quantification of digested proteins has been published .…”

Quantification of intact covalently metal labeled proteins using ESI-MS/MS

“…If this condition is met, absolute quantification can be achieved for proteins and peptides, and when isotope dilution is applied, the data become very accurate . Some time ago, we observed characteristic fragments of DOTA derivative‐labeled peptides in electrospray ionization (ESI) spectra (and others in matrix‐assisted laser desorption/ionization spectra as well), which allowed quantification of peptides, as we could demonstrate in a first, orientating study . We could validate the reliability of this approach by ICP‐MS.…”