Design and Synthesis of Visible Isotope-Coded Affinity Tags for the Absolute Quantification of Specific Proteins in Complex Mixtures

Abstract

Identification of proteins in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained. We recently introduced isotope-coded affinity tags (ICAT reagents) for the relative quantification of proteins present in two or more biological samples. In this report, we describe a new generation of ICAT reagents that contain the following additional features: (1) a visible tag that allows the electrophoretic position of tagged peptides during separation to be easily monitored; (2) a photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis; (3) an isotope tag that contains carbon-13 and nitrogen-15 atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptides by reverse-phase HPLC. These reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residues. Peptide modification chemistry is also reported that allows tagging of peptides that are devoid of cysteine. The synthesis of these visible isotope-coded affinity tags (VICAT reagents), and their reaction with peptides are described in this report. VICAT reagents containing a carbon-14 visible probe or an NBD fluorophore are described. These reagents are most useful for the determination of the absolute quantity of specific target proteins in complex protein mixtures such as serum or cell lysates.

Training in metabolomics research. I. Designing the experiment, collecting and extracting samples and generating metabolomics data

“…[76] The initial form of the reagent was labeled with deuterium ( 2 H 8 ) but was replaced by the 13 C 8 form. [77] The best way to avoid problems created by the chromatographic differences isotopically labeled and unlabeled metabolites is to compare the areas under each peak rather than to use the ratio of their intensities. There is also a risk with deuterated standards in that many are introduced by exchange reactions.…”

Affinity Chromatography: Principles and Applications

“…Biotin is also used in isotopically coded affinity tags (ICATs) which can be used to compare the protein content in two different samples (Bottari et al, 2004). The ICAT consists of two labels, one which contains deuterium (heavy) and one which contains only hydrogen (light).…”

Perspective: A Program to Improve Protein Biomarker Discovery for Cancer

“…25 Anti-peptide antibodies will be useful for enrichment in the SISCAPA approach, 26 and appropriate isotopically labeled peptide reagents will be needed.…”