Enrichment of N-terminal cysteinyl-peptides by covalent capture

Giron, Priscille; Dayon, Loı̈c; David, Fabrice; Sanchez, Jean‐Charles; Rose, Keith

doi:10.1016/j.jprot.2008.11.009

Cited by 14 publications

(31 citation statements)

References 29 publications

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“…In addition, comprehensive sequence analysis of the human genome indicates that the occurrence of cysteine at the N-termini is markedly suppressed,32 and none of the forty most abundant proteins in plasma and heart tissue we examined have cysteine at their N-termini, perhaps to avoid reactions with biological aldehydes. A similar finding was reported recently, i.e., only 0.5% (99 out of 20,327) of human proteins are predicted to have an N-terminal cysteine 30. Furthermore, it is estimated that N-terminal amines in over 80% of all mammalian proteins are acetylated, and consequently, will not react with aldehydes.…”

Section: Resultssupporting

confidence: 87%

Chemical Methods for the Detection of Protein N-Homocysteinylation via Selective Reactions with Aldehydes

et al. 2009

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Elevated blood levels of homocysteine (Hcy), hyperhomocysteinemia or homocystinuria, have been associated with various diseases and conditions. Homocysteine thiolactone (Hcy TL) is a metabolite of Hcy and reacts with amine groups in proteins to form stable amides, homocystamides or N-homocysteinylated proteins. It has been proposed that protein N-homocysteinylation contributes to the cytotoxicity of elevated Hcy. Due to its heterogeneity and relatively low abundance, detection of this post-translational modification remains challenging. On the other hand, the gamma-aminothiol group in homocystamides imparts different chemical reactivities than the native proteins. Under mildly acidic conditions, gamma-aminothiols irreversibly and stoichiometrically react with aldehydes to form stable 1,3-thiazines, whereas the reversible Schiff base formation between aldehydes and amino groups in native proteins is markedly disfavored due to protonation of amines. As such, we have developed highly selective chemical methods to derivatize N-homocysteinylated proteins with various aldehyde tags, thereby facilitating the subsequent analyses. For instance, fluorescent or biotin tagging coupled with gel electrophoresis permits quantification and global profiling of complex biological samples, such as hemoglobin and plasma from rat, mouse and human; affinity enrichment with aldehyde resins drastically reduces sample complexity. In addition, different reactivities of lysine residues in hemoglobin towards Hcy TL were observed.

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Section: Resultssupporting

confidence: 87%

Chemical Methods for the Detection of Protein N-Homocysteinylation via Selective Reactions with Aldehydes

et al. 2009

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“…The peptides and proteins that contain or do not contain cysteine are displayed. Unpublished data, from Giron et al (2009a). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com] CYSTEINE-TAGGING STRATEGIES & take place on the proteins or on the peptides without interfering with the bottom-up analysis.…”

Section: B Chemistry and Modification Of Sulfhydryl Groups In Proteomentioning

confidence: 99%

“…Originally developed by Rose's group to purify synthetic peptides, the CC was recently exploited for proteomics. In a proof-of-principle study, CC reduced sample complexity and enriched the Nterminal cysteinyl tryptic peptides from standard proteins with MS and MS/MS analyses (Giron et al, 2009a). The strategy was recently extended to all cysteine-containing peptides with cysteinyl tags; namely, cysteine-reactive covalent-capture tags (C 3 T) (Giron et al, 2009b).…”

Section: Covalent Chromatographymentioning

confidence: 99%

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Cysteine tagging for MS‐based proteomics

Giron

Dayon

Sanchez

2010

Mass Spectrometry Reviews

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Amino acid-tagging strategies are widespread in proteomics. Because of the central role of mass spectrometry (MS) as a detection technique in protein sciences, the term "mass tagging" was coined to describe the attachment of a label, which serves MS analysis and/or adds analytical value to the measurements. These so-called mass tags can be used for separation, enrichment, detection, and quantitation of peptides and proteins. In this context, cysteine is a frequent target for modifications because the thiol function can react specifically by nucleophilic substitution or addition. Furthermore, cysteines present natural modifications of biological importance and a low occurrence in the proteome that justify the development of strategies to specifically target them in peptides or proteins. In the present review, the mass-tagging methods directed to cysteine residues are comprehensively discussed, and the advantages and drawbacks of these strategies are addressed. Some concrete applications are given to underline the relevance of cysteine-tagging techniques for MS-based proteomics.

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“…Researchers have also dealt with enrichment based on targeting protein Nterminal peptides [8] and C-terminal peptides [9]. Even the presence of a specific amino acid at the peptide N-terminus has been utilized [10]. Most tagging methods allow for specific enrichment of peptides containing a particular amino acid such as tryptophan [11], methionine [12], arginine or histidine [13].…”

Section: Introductionmentioning

confidence: 99%