Synthesis and Self-Alkylation of Isotope-Coded Affinity Tag Reagents

Zhang, Zhidong; Edwards, Patrick J. B.; Roeske, Roger W.; Guo, Lili

doi:10.1021/bc049777y

Cited by 5 publications

(3 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Besides, Ratner and Clarke showed that iodoacetic acid is able to alkylate the thioproline and that it results in the opening of the thiazolidine ring 18. This self‐alkylation side reaction has also been observed in standard Cys‐tagging techniques such as ICAT 20. When using the Boc‐ITC, it was found that this side reaction was prevented, since such side products were almost absent.…”

Section: Resultsmentioning

confidence: 84%

Cysteine‐reactive covalent capture tags for enrichment of cysteine‐containing peptides

Giron

Dayon

Mihala

et al. 2009

Rapid Comm Mass Spectrometry

View full text Add to dashboard Cite

Considering the tremendous complexity and the wide dynamic range of protein samples from biological origin and their proteolytic peptide mixtures, proteomics largely requires simplification strategies. One common approach to reduce sample complexity is to target a particular amino acid in proteins or peptides, such as cysteine (Cys), with chemical tags in order to reduce the analysis to a subset of the whole proteome. The present work describes the synthesis and the use of two new cysteinyl tags, so-called cysteine-reactive covalent capture tags (C3T), for the isolation of Cys-containing peptides. These bifunctional molecules were specifically designed to react with cysteines through iodoacetyl and acryloyl moieties and permit efficient selection of the tagged peptides. To do so, a thioproline was chosen as the isolating group to form, after a deprotection/activation step, a thiazolidine with an aldehyde resin by the covalent capture (CC) method. The applicability of the enrichment strategy was demonstrated on small synthetic peptides as well as on peptides derived from digested proteins. Mass spectrometric (MS) analysis and tandem mass spectrometric (MS/MS) sequencing confirmed the efficient and straightforward selection of the cysteine-containing peptides. The combination of C3T and CC methods provides an effective alternative to reduce sample complexity and access low abundance proteins.

show abstract

Section: Resultsmentioning

confidence: 84%

Cysteine‐reactive covalent capture tags for enrichment of cysteine‐containing peptides

Giron

Dayon

Mihala

et al. 2009

Rapid Comm Mass Spectrometry

View full text Add to dashboard Cite

show abstract

“…The synthesis of the diamine component 4 was completed following a literature procedure (Scheme 2) (28,29). Thus, two molar equivalents of diethylene glycol 7 were added in a 1,4addtion reaction to acrylonitrile.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of Acid-Cleavable Light Isotope-Coded Affinity Tags (ICAT-L) for Potential Use in Proteomic Expression Profiling Analysis

et al. 2005

View full text Add to dashboard Cite

A convenient synthesis of some homologous light isotope-coded affinity tags (ICAT-L) containing an acid-labile moiety between the affinity component biotin and an electrophilic polar linker is described. These light ICAT reagents give smooth mass spectral signals in tandem mass spectrometry (MS/MS) analyses of some commercially available cysteine-containing peptides. However, these ICAT molecules are designed for use in identification and relative quantification of whole or partially purified cellular and tissue proteomes. Since the biotin moiety can be readily cleaved off the reagent after mass tagging, undesired residual fragmentation patterns caused by biotin of derived peptides, as normally observed using biotin-containing ICAT reagents, are effectively eliminated. This strategy should enhance peptide sequence coverage significantly which, in turn, should result in improving the quality of data obtained during data-dependent peptide mass and tandem mass spectral analysis of whole proteomes.

show abstract

“…Protein purification and its detection/localization are both essential components of proteomics and biomolecular research − . Proteins are routinely purified utilizing affinity purification strategies with either chemical or biological tags − . For efficient purification, either peptides or proteins are used as affinity tags − .…”

Section: Introductionmentioning

confidence: 99%

Dual Function Labeling of Biomolecules Based on DsRed-Monomer

et al. 2008

View full text Add to dashboard Cite

Unavailability of fusion tags that possess both affinity and visualization properties is a hurdle for biomolecular research. Typically, either a choice is made between an affinity tag and a reporter tag or both are employed in tandem if a fusion can be made at both termini of the target biomolecule. In this work, we have developed a site-specific genetic fusion approach employing DsRed-Monomer, a red fluorescent protein, that provides for both affinity and reporter functionality in a single tag. As a proof-of-concept, two fusion proteins, bradykinin-DsRed-Monomer and calmodulin-DsRed-Monomer, were prepared for the study. These fusion proteins were purified using a copper-immobilized column based on the inherent copper-binding property of DsRed-Monomer. Spectroscopic characterization of fusion proteins and comparison with native DsRed-Monomer showed no effect of fusion on the properties of DsRed-Monomer. Further, bradykinin-DsRed-Monomer was employed in the development of a competitive fluorescence immunoassay for the peptide bradykinin. Calmodulin-DsRed-Monomer was used to detect binding of the calmodulin ligand chlorpromazine, based on a change in the fluorescence of DsRed-Monomer upon binding of chlorpromazine to calmodulin. The studies performed demonstrate the application of DsRed-Monomer as a dual function tag indicating the potential usefulness of DsRed-monomer in proteomics and biomolecular research.

show abstract

Synthesis and Self-Alkylation of Isotope-Coded Affinity Tag Reagents

Cited by 5 publications

References 25 publications

Cysteine‐reactive covalent capture tags for enrichment of cysteine‐containing peptides

Cysteine‐reactive covalent capture tags for enrichment of cysteine‐containing peptides

Synthesis of Acid-Cleavable Light Isotope-Coded Affinity Tags (ICAT-L) for Potential Use in Proteomic Expression Profiling Analysis

Dual Function Labeling of Biomolecules Based on DsRed-Monomer

Contact Info

Product

Resources

About