This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Method Article
Characterization of membrane protein interactomes by Co-interacting Protein Identification Technology (CoPIT)
Sandra Pankow
Yates lab, The Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla, CA 92037
Corresponding Author
John R Yates
Yates lab, The Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla, CA 92037
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Finding interaction partners of proteins is an important step in understanding their function. While co-immunoprecipitation of proteins followed by mass spectrometric identification (AP-MS) has become a standard approach to identify interaction partners, characterization of membrane protein interactomes is still challenging because of the typically low abundance and high hydrophobicity of membrane proteins. Here, we describe CoPIT (for Co-interacting Protein Identification Technology), an approach to comprehensively identify and characterize the interactome of membrane proteins. The CoPIT protocol includes experimental and computational methods to establish membrane protein interactome networks and to monitor interactome changes upon perturbation. While the approach in particular improves membrane protein interactome analysis as demonstrated by using a low abundant membrane protein (Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)) as a model, it is applicable to all types of proteins. The time required to complete the protocol varies from 3 days to several weeks depending on whether all or only part of it is carried out.
Keywords
protein complex, protein interactions, network dynamics, ion channel, cystic fibrosis, immunoprecipitation, deep interactome
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.docx
Table 1 Troubleshooting
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 30 Nov, 2015
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biochemistry
Associated Publications
∆F508 CFTR interactome remodelling promotes rescue of cystic fibrosis
by Sandra Pankow, Casimir Bamberger, Diego Calzolari, +3
Nature (30 November, 2015)
Learn more about our company.
This is a preprint. It has not completed peer review.
Method Article
Characterization of membrane protein interactomes by Co-interacting Protein Identification Technology (CoPIT)
Sandra Pankow
Yates lab, The Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla, CA 92037
Corresponding Author
John R Yates
Yates lab, The Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla, CA 92037
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 30 Nov, 2015
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biochemistry
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Finding interaction partners of proteins is an important step in understanding their function. While co-immunoprecipitation of proteins followed by mass spectrometric identification (AP-MS) has become a standard approach to identify interaction partners, characterization of membrane protein interactomes is still challenging because of the typically low abundance and high hydrophobicity of membrane proteins. Here, we describe CoPIT (for Co-interacting Protein Identification Technology), an approach to comprehensively identify and characterize the interactome of membrane proteins. The CoPIT protocol includes experimental and computational methods to establish membrane protein interactome networks and to monitor interactome changes upon perturbation. While the approach in particular improves membrane protein interactome analysis as demonstrated by using a low abundant membrane protein (Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)) as a model, it is applicable to all types of proteins. The time required to complete the protocol varies from 3 days to several weeks depending on whether all or only part of it is carried out.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Associated Publications
∆F508 CFTR interactome remodelling promotes rescue of cystic fibrosis
by Sandra Pankow, Casimir Bamberger, Diego Calzolari, +3
Nature (30 November, 2015)