Dataset Information

Biotin tagging coupled with amino acid-coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells.

ABSTRACT: In mammalian cells, when tandem affinity purification approach is employed, the existence of untagged endogenous target protein and repetitive washing steps together result in overall low yield of purified/stable complexes and the loss of weakly and transiently interacting partners of biological significance. To avoid the trade-offs involving in methodological sensitivity, precision, and throughput, here we introduce an integrated method, biotin tagging coupled with amino acid-coded mass tagging, for highly sensitive and accurate screening of mammalian protein-protein interactions. Without the need of establishing a stable cell line, using a short peptide tag which could be specifically biotinylated in vivo, the biotin-tagged target/bait protein was then isolated along with its associates efficiently by streptavidin magnetic microbeads in a single step. In a pulled-down complex amino acid-coded mass tagging serves as "in-spectra" quantitative markers to distinguish those bait-specific interactors from non-specific background proteins under stringent criteria. Applying this biotin tagging coupled with amino acid-coded mass tagging approach, we first biotin-tagged in vivo a multi-functional protein family member, 14-3-3epsilon, which was expressed at close to endogenous level. Starting with approximately 20 millions of 293T cells which were significantly less than what needed for a tandem affinity purification run, 266 specific interactors of 14-3-3epsilon were identified in high confidence.

SUBMITTER: He YF

PROVIDER: S-EPMC4302342 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Biotin tagging coupled with amino acid-coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells.

He Yu-Fei YF Bao Hui-Min HM Xiao Xiao-Feng XF Zuo Shuai S Du Ru-Yun RY Tang Si-Wei SW Yang Peng-Yuan PY Chen Xian X

Proteomics 20091201 24

In mammalian cells, when tandem affinity purification approach is employed, the existence of untagged endogenous target protein and repetitive washing steps together result in overall low yield of purified/stable complexes and the loss of weakly and transiently interacting partners of biological significance. To avoid the trade-offs involving in methodological sensitivity, precision, and throughput, here we introduce an integrated method, biotin tagging coupled with amino acid-coded mass tagging ...[more]

PMID: 19834888

Similar Datasets

Project description:BackgroundMethods used to quantify protein from biological samples are often inaccurate with significant variability that requires care to minimize. The errors result from losses during protein preparation and purification and false detection of interfering compounds or elements. Amino acid analysis (AAA) involves a series of chromatographic techniques that can be used to measure protein levels, avoiding some difficulties and providing specific compositional information. However, unstable derivatives, that are toxic and can be costly, incomplete reactions, inadequate chromatographic separations, and the lack of a single hydrolysis method with sufficient recovery of all amino acids hinder precise protein quantitation using AAA.ResultsIn this study, a hydrophilic interaction chromatography based method was used to separate all proteinogenic amino acids, including isobaric compounds leucine and isoleucine, prior to detection by multiple reaction monitoring with LC-MS/MS. Through inclusion of commercially available isotopically labeled (13C, 15N) amino acids as internal standards we adapted an isotopic dilution strategy for amino acid-based quantification of proteins. Three hydrolysis methods were tested with ubiquitin, bovine serum albumin, (BSA), and a soy protein biological reference material (SRM 3234; NIST) resulting in protein estimates that were 86-103%, 82-94%, and 90-99% accurate for the three protein samples respectively. The methane sulfonic acid hydrolysis approach provided the best recovery of labile amino acids including: cysteine, methionine and tryptophan that are challenging to accurately quantify.ConclusionsAccurate determination of protein quantity and amino acid composition in heterogeneous biological samples is non-trivial. Recent advances in chromatographic phases and LC-MS/MS based methods, along with the availability of isotopic standards can minimize difficulties in analysis and improve protein quantitation. A robust method is described for high-throughput protein quantification and amino acid compositional analysis. Since accurate measurement of protein quality and quantity are a requirement for many biological studies that relate to crop improvement or more generally, our understanding of metabolism in living systems, we envision this method will have broad applicability.

Dataset Information

Biotin tagging coupled with amino acid-coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells.

Publications

Biotin tagging coupled with amino acid-coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets