G2C::Proteomics

Targeted tandem affinity purification of PSD-95 recovers core
postsynaptic complexes and schizophrenia suscepibility proteins

Esperanza Fernandez, Mark O Collins, Rachel T Uren, Maksym V Kopanitsa, Noboru H Komiyama,
Mike DR Croning, Lysimachos Zografos, J Douglas Armstrong, Jyoti S Choudhary and Seth GN Grant

Click to enlarge

FAQs

1. What is the Tandem Affinity Purification (TAP) method?
2. What advantages does the TAP method offer?
3. Which proteins belong to the core set of postsynaptic proteins?
4. Has this protein been identified in any PSD purification and/or in the MASC complex, PSD or other neuronal complex before?
5. Does this protein directly bind PSD-95?
6. What is the G2C database?
7. Is this gene related to any disorder?


1. What is the Tandem Affinity Purification (TAP) method?

The Tandem Affinity Purification method consists of a genetic modification of the protein of interest by fusion with a TAP (two different tags separated by a protease site) tag into the C- or N- terminal end of the protein (Rigaut et al., 1999). The tagged protein is isolated (with its associated proteins) in a tandem procedure: a first purification is followed by a protease cleavage and the complex is recovered in a second purification that ends with the elution in native conditions.

2. What advantages does the TAP method offer?

The TAP method allows a more stringent purification removing weak interactors and potential contaminants. The TAP method also has technical advantages over immunoprecipitation that is limited by i) the availability of suitable antibodies and their cross reaction with other proteins, ii) the possibility that the antibody-protein interaction is affected by either posttranslational modifications or by the binding with other proteins, iii) the antibody may disrupt interacting partners, and iv) the harsh conditions for the complex elution may result in protein degradation.

3. Which proteins belong to the core set of postsynaptic proteins?

lists the proteins identified in at least three of four Tandem Affinity Purifications that are considered as the core of the postsynaptic terminal of excitatory synapses. Table 2 shows the synaptic proteins that have been purified once or twice and could be weak or non-abundant PSD-95 interactors. These proteins should not be considered as part of the PSD-95 core complex.

4. Has this protein been identified in any PSD purification and/or in the MASC complex, PSD or other neuronal complex before?

Go to Supplementary Table 1 and identify the gene name or the protein ID. Follow the G2Cdb link and the G2Cdb gene report is shown. Other gene lists containing this gene are reported.

5. Does this protein directly bind PSD-95?

PSD-95 binary interactors that belong to the PSD-95 complex are listed in Supplementary Table 3. Literature reference are provided directly to PubMed for inspection.

6. What is the G2C database?

G2Cdb is an integrated database for neurobiologists that contains data linking gene function to cognition. The datasets include gene and protein expression, synaptic physiology, mouse and human behaviour data with synapse proteomics. The datasets includes that generated by members of the Genes to Cognition program and literature mined data.

7. Is this gene related to any disorder?

PSD-95 interactors involved in mental disease are shown in the Table 3. Following the G2Cdb ID in the Supplementary Table 1 you have access to the G2Cdb gene report that links to behavioural and physiological data in knockout mice.

References

Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. and Seraphin, B. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol, 17, 1030-1032.