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Related Concept Videos

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Tagging 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...
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Peptide 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...

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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
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Absolute quantification of specific proteins in complex mixtures using visible isotope-coded affinity tags.

Yu Lu1, Patricia Bottari, Ruedi Aebersold

  • 1Department of Chemistry, University of Washington, Seattle, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 9, 2007
PubMed
Summary

Researchers developed Visible Isotope-Coded Affinity Tags (VICAT reagents) for precise protein quantification in complex samples. This method enables accurate measurement of protein abundance, aiding disease biomarker discovery and analysis of biological samples.

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Accurate determination of absolute protein abundance is crucial for disease biomarker discovery and understanding biological systems.
  • Existing methods often face challenges in quantifying specific proteins within complex biological mixtures like cell lysates or serum.

Purpose of the Study:

  • To develop a novel set of reagents for the absolute quantification of target proteins in complex mixtures.
  • To establish a methodology utilizing these reagents for sensitive and specific protein detection and measurement.

Main Methods:

  • Development of Visible Isotope-Coded Affinity Tags (VICAT reagents) incorporating cysteine reactivity, visible probes, photo-releasable biotin, and heavy isotope tags.
  • Integration of VICAT reagents with isoelectric focusing and reverse-phase microbore chromatography coupled with tandem mass spectrometry (LC-MS/MS).
  • Application of the method to determine the absolute abundance of target proteins in complex samples, such as cell lysates.

Main Results:

  • Successful development and characterization of VICAT reagents for comprehensive protein tagging and analysis.
  • Demonstration of the method's capability to accurately quantify absolute protein abundance in complex biological mixtures.
  • Validation of the approach for differentiating target peptides from internal standards using heavy isotope tags.

Conclusions:

  • VICAT reagents provide a powerful tool for the absolute quantification of proteins in complex mixtures.
  • This methodology holds significant potential for advancing disease biomarker discovery and analyzing low-abundance proteins in biological fluids.
  • The approach is also adaptable for detecting post-translational modifications and protein splice variants.