Dataset Information

Peptide level immunoaffinity enrichment enhances ubiquitination site identification on individual proteins.

ABSTRACT: Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins of interest, including site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). Recent publications have also highlighted the use of peptide-level immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for global characterization of ubiquitination sites. Here we investigated the utility of this technique for focused mapping of ubiquitination sites on individual proteins. For a series of membrane-associated and cytoplasmic substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor ? (TCR?), we observed that K-GG peptide immunoaffinity enrichment consistently yielded additional ubiquitination sites beyond those identified in protein level AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were prepared and used to compare the abundances of individual K-GG peptides from samples prepared in parallel. Consistently, K-GG peptide immunoaffinity enrichment yielded greater than fourfold higher levels of modified peptides than AP-MS approaches. Using this approach, we went on to characterize inducible ubiquitination on multiple members of the T-cell receptor complex that are functionally affected by endoplasmic reticulum (ER) stress. Together, these data demonstrate the utility of immunoaffinity peptide enrichment for single protein ubiquitination site analysis and provide insights into the ubiquitination of HER2, DVL2, and proteins in the T-cell receptor complex.

SUBMITTER: Anania VG

PROVIDER: S-EPMC3879610 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Peptide level immunoaffinity enrichment enhances ubiquitination site identification on individual proteins.

Anania Veronica G VG Pham Victoria C VC Huang XiaoDong X Masselot Alexandre A Lill Jennie R JR Kirkpatrick Donald S DS

Molecular & cellular proteomics : MCP 20131018 1

Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins ...[more]

PMID: 24142993

Similar Datasets

Project description:Oral cancer is one of the most common cancers worldwide, and there are currently no biomarkers approved for aiding its management. Although many potential oral cancer biomarkers have been discovered, very few have been verified in body fluid specimens in parallel to evaluate their clinical utility. The lack of appropriate multiplexed assays for chosen targets represents one of the bottlenecks to achieving this goal. In the present study, we develop a peptide immunoaffinity enrichment-coupled multiple reaction monitoring-mass spectrometry (SISCAPA-MRM) assay for verifying multiple reported oral cancer biomarkers in saliva. We successfully produced 363 clones of mouse anti-peptide monoclonal antibodies (mAbs) against 36 of 49 selected targets, and characterized useful mAbs against 24 targets in terms of their binding affinity for peptide antigens and immuno-capture ability. Comparative analyses revealed that an equilibrium dissociation constant (KD ) cut-off value < 2.82 × 10-9 m could identify most clones with an immuno-capture recovery rate >5%. Using these mAbs, we assembled a 24-plex SISCAPA-MRM assay and optimized assay conditions in a 25-μg saliva matrix background. This multiplexed assay showed reasonable precision (median coefficient of variation, 7.16 to 32.09%), with lower limits of quantitation (LLOQ) of <10, 10-50, and >50 ng/ml for 14, 7 and 3 targets, respectively. When applied to a model saliva sample pooled from oral cancer patients, this assay could detect 19 targets at higher salivary levels than their LLOQs. Finally, we demonstrated the utility of this assay for quantification of multiple targets in individual saliva samples (20 healthy donors and 21 oral cancer patients), showing that levels of six targets were significantly altered in cancer compared with the control group. We propose that this assay could be used in future studies to compare the clinical utility of multiple oral cancer biomarker candidates in a large cohort of saliva samples.

Dataset Information

Peptide level immunoaffinity enrichment enhances ubiquitination site identification on individual proteins.

Publications

Peptide level immunoaffinity enrichment enhances ubiquitination site identification on individual proteins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets