Metal labeling of biopolymers for high precision quantification

Autor*innen

• Yide He

Parameter

• Lehrbücher
• Universitätslehrbücher

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To address challenges in quantitative proteomics, Metal-coded affinity tagging (MeCAT) was developed for sensitive quantification of proteins and peptides. This method labels biopolymers with lanthanide ions in DOTA complexes instead of isotopes. Various MeCATs have been successfully used to quantify peptides and proteins; however, the large size of DOTA complexes can hinder access to some active sites, limiting broader application. This book introduces a new MeCAT label based on in situ Click Chemistry (MeCAT-Click), which enhances labeling efficiency and reduces steric hindrance. The label is compatible with ESI-MS techniques, enabling clear identification of labeled peptides and proteins. Multiple analytical approaches for MeCAT-Click labeled samples include liquid chromatography and gel electrophoresis, followed by mineralization and direct infusion ICP-MS, applicable to both intact and digested proteins. Beyond standard samples, it was used to quantify proteins during heat shock in Escherichia coli, demonstrating its effectiveness in real samples. Two parallel experimental approaches combined elemental and molecular MS techniques complementarily. The quantification of peptides labeled with MeCAT-Click was validated using characteristic fragments in ESI-MS/MS, with results consistent with ICP-MS and full scan spectra from ESI-MS. The concept of peptide quantification using elemental reporter ions was also confirmed.

• Sprache: Englisch
• Erscheinungsdatum: 2015