Enrichment of N-terminal cysteinyl-peptides by covalent capture.
The detection of low abundance proteins in complex biological samples is still a challenge in proteomics. To circumvent this obstacle a number of strategies involving the targeting of subsets of proteins or peptides were developed. The following work describes a new approach to simplify peptide mixtures by enrichment of N-terminal cysteinyl peptides (and to some extent N-terminal threonine peptides). The strategy is based on the use of an isolation method, so-called covalent capture (CC), which relies on the formation of a covalent bond between an N-terminal free cysteine or N-terminal free threonine and an aldehyde fixed on a solid support. The CC is highly selective. It permits extensive washes of the resin for the elimination of non-specific moieties before the release of the captured peptides. The application of the CC to proteomics was evaluated on tryptic peptides of standard proteins and test protein mixtures. The procedure demonstrated a significant reduction in sample complexity, while allowing the identification of N-terminal cysteinyl peptides hidden in the non-fractionated samples. This new strategy provides an efficient tool to existing proteomics approaches to reduce sample complexity and potentially identify less abundance proteins.
Giron
Priscille
P
K. Rose's Group, Department of Structural Biology and Bioinformatics, CH-1211 Geneva 4, Switzerland.
Dayon
Loïc
L
David
Fabrice
F
Sanchez
Jean-Charles
JC
Rose
Keith
K
D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D013912:Threonine / Q000378:metabolism*
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D003545:Cysteine