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Chemoenzymatic labeling of protein C-termini for positive selection of C-terminal peptides.

ABSTRACT: Many proteomic experiments require selective labeling of either N- or C-termini of proteins and recovery of terminal peptides. Although N-termini can be selectively labeled, selective labeling of protein C-termini has not been possible due to the difficulty in discriminating between the carboxyl group on the C-terminus versus that on aspartate and glutamate residues. Here we describe the first simple proteomic approach for positive selection of protein C-termini, Profiling Protein C-Termini by Enzymatic Labeling (ProC-TEL). ProC-TEL uses carboxypeptidase Y and other readily available reagents to selectively add an affinity tag to protein C-termini and to capture C-terminal peptides from complex cell lysates for mass spectrometry (MS) identification. Using ProC-TEL, we identify novel C-terminal processing and internal proteolytic cleavage events. These results indicate that ProC-TEL provides a straightforward approach for profiling C-terminal peptides and identifying protein processing in complex biological samples.

SUBMITTER: Xu G

PROVIDER: S-EPMC3199308 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Chemoenzymatic labeling of protein C-termini for positive selection of C-terminal peptides.

Xu Guoqiang G Shin Sung Bin Y SB Jaffrey Samie R SR

ACS chemical biology 20110810 10

Many proteomic experiments require selective labeling of either N- or C-termini of proteins and recovery of terminal peptides. Although N-termini can be selectively labeled, selective labeling of protein C-termini has not been possible due to the difficulty in discriminating between the carboxyl group on the C-terminus versus that on aspartate and glutamate residues. Here we describe the first simple proteomic approach for positive selection of protein C-termini, Profiling Protein C-Termini by E ...[more]

PMID: 21812462

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Project description:Site-specific chemical modification of proteins is important for many applications in biology and biotechnology. Recently, our laboratory and others have exploited the high specificity of the enzyme protein farnesyltransferase (PFTase) to site-specifically modify proteins through the use of alternative substrates that incorporate bioorthogonal functionality including azides and alkynes. In this study, we evaluate two aldehyde-containing molecules as substrates for PFTase and as reactants in both oxime and hydrazone formation. Using green fluorescent protein (GFP) as a model system, we demonstrate that the purified protein can be enzymatically modified with either analogue to yield aldehyde-functionalized proteins. Oxime or hydrazone formation was then employed to immobilize, fluorescently label, or PEGylate the resulting aldehyde-containing proteins. Immobilization via hydrazone formation was also shown to be reversible via transoximization with a fluorescent alkoxyamine. After characterizing this labeling strategy using pure protein, the specificity of the enzymatic process was used to selectively label GFP present in crude E. coli extract followed by capture of the aldehyde-modified protein using hydrazide-agarose. Subsequent incubation of the immobilized protein using a fluorescently labeled or PEGylated alkoxyamine resulted in the release of pure GFP containing the desired site-specific covalent modifications. This procedure was also employed to produce PEGylated glucose-dependent insulinotropic polypeptide (GIP), a protein with potential therapeutic activity for diabetes. Given the specificity of the PFTase-catalyzed reaction coupled with the ability to introduce a CAAX-box recognition sequence onto almost any protein, this method shows great potential as a general approach for selective immobilization and labeling of recombinant proteins present in crude cellular extract without prior purification. Beyond generating site-specifically modified proteins, this approach for polypeptide modification could be particularly useful for large-scale production of protein conjugates for therapeutic or industrial applications.

Dataset Information

Chemoenzymatic labeling of protein C-termini for positive selection of C-terminal peptides.

Publications

Chemoenzymatic labeling of protein C-termini for positive selection of C-terminal peptides.

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