Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions
Open accessAbstract
Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC-green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.
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High protein ratios in the range of several orders of magnitude typically occur in interaction proteomics experiments, where early mixing of isotope-labeled samples is usually not possible.
“Such ratios typically occur in the context of interaction proteomics experiments (56), where early mixing of isotope-labeled samples is usually not possible and some of the principal advantages of metabolic labeling are therefore lost.”
From Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ (2014) · cites this paper as [56]
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- Authors
- Hubner, N. C., Bird, A. W., Cox, J., Splettstoesser, B., Bandilla, P., Poser, I., Hyman, A., & Mann, M.
- Affiliations
- Max Planck Institute of Biochemistry (DE)
- Max Planck Institute of Molecular Cell Biology and Genetics (DE)
- Corresponding author
- Hyman, Anthony
- Mann, Matthias
- Venue
- Journal of Cell Biology
- Publisher
- Rockefeller University Press
- Published
- 2010-05-17
- Volume
- 189
- Issue
- 4
- Pages
- 739-754
- ISSN
- 0021-9525 (ISSN-L)
- 1540-8140
- PMC
- PMC2872919
- OpenAlex
- W2059041864
- Cited by
- 440
- References
- 67
- Citations by year
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- FWCI
- 18.51
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by-nc-sa
- Locations
- The Journal of Cell Biology (publishedVersion, cc-by-nc-sa)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
- PubMed Central (publishedVersion, cc-by-nc-sa)
- PubMed (publishedVersion)
- Max Planck Institute for Plasma Physics (submittedVersion)
- Max Planck Digital Library (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Biotin and Related Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bioinformatics and Genomic Networks
- Keywords
- protein-protein interaction, QUBIC, anaphase-promoting complex, pericentrin, TACC3, mitotic spindle, drug-induced perturbation, protein isoforms
- MeSH terms
- Antigens, Fluorescent Antibody Technique, Humans, Models, Biological, Phosphorylation, Transgenes, Chromosomes, Artificial, Bacterial, Genomics, Protein Interaction Mapping, Proteomics
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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