Related Experiment Video
Updated: Jun 22, 2026
07:19
Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System
Published on: April 26, 2024
Fragmentation behavior of metal-coded affinity tag (MeCAT)-labeled peptides
Stefan Pieper1, Sebastian Beck, Robert Ahrends
1Department of Chemistry, Humboldt-Universitaet zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany.
Rapid Communications in Mass Spectrometry : RCM
|June 9, 2009
Summary
Metal-coded affinity tagging (MeCAT) enables quantitative proteomics. This study details MeCAT-labeled peptide fragmentation under CID, ECD, and IRMPD, aiding structural elucidation and identification of labeled compounds.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Quantitative proteomics is crucial in life sciences, with increasing relevance in protein quantification.
- Metal-coded affinity tagging (MeCAT) offers tools for both relative and absolute quantification.
- Understanding fragmentation behavior of modified peptides is essential for structural elucidation.
Purpose of the Study:
- To investigate the fragmentation behavior of MeCAT-labeled peptides under different dissociation conditions (CID, ECD, IRMPD).
- To assess the utility of these fragmentation methods for sequence elucidation and identification of MeCAT-labeled compounds.
- To characterize by-products from the MeCAT labeling process and optimize reaction conditions.
Main Methods:
- Analysis of MeCAT-labeled peptides using Collision Induced Dissociation (CID).
- Analysis of MeCAT-labeled peptides using Electron Capture Dissociation (ECD).
- Analysis of MeCAT-labeled peptides using Infrared Multiphoton Dissociation (IRMPD).
- Structural characterization of labeling by-products.
Main Results:
- CID and ECD facilitated straightforward sequence elucidation of MeCAT-labeled peptides.
- IRMPD produced characteristic fragments from the tagging group, enabling screening for labeled compounds.
- Side reactions during labeling were investigated, by-products characterized, and conditions optimized to minimize their formation.
Conclusions:
- Fragmentation analysis of MeCAT-labeled peptides using CID, ECD, and IRMPD provides valuable insights for structural elucidation and quantification.
- IRMPD serves as an effective screening method for identifying MeCAT-labeled peptides.
- Optimization of labeling conditions reduces by-product formation, enhancing the reliability of MeCAT in quantitative proteomics.
Related Concept Videos
01:24Tagging and Fusion Proteins
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
01:01Mass Spectrometry: Carboxylic Acid, Ester, and Amide Fragmentation
The fragmentation patterns observed for compounds such as carboxylic acids, esters, and amides in the mass spectra include ⍺-cleavage and McLafferty rearrangement. Fragmentation by ⍺-cleavage preferentially occurs at the carbon-carbon bond at the ⍺-position next to the carboxylic group to generate a neutral radical and a cation. Long chain compounds with hydrogen at their γ-carbon undergo McLafferty rearrangement to give a radical cation and a neutral alkene.
For example, the fragmentation of...
For example, the fragmentation of...
01:33Peptide Identification Using Tandem Mass Spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
01:20Mass Spectrometry: Molecular Fragmentation Overview
The ionization of a molecule into a molecular ion inside the mass spectrometer causes instability in the molecule's structure due to the loss of an electron. This eventually leads to the fragmentation or breaking of some bonds in the molecule. The fragmentation occurs predominantly at specific bonds to yield relatively stable fragments.
One type of fragmentation pattern is the cleavage of a single bond in the molecular ion. The cleavage leads to a radical and a cation. The cleavage can occur at...
One type of fragmentation pattern is the cleavage of a single bond in the molecular ion. The cleavage leads to a radical and a cation. The cleavage can occur at...