This research was supported by grants of the Deutsche Forschungsgemeinschaft GraKo1121, Hu1011/2-1 and SFB940 to S.H.

Note: Detailed information regarding buffers indicated in the protocol is available in Table 1.
1. Fixation of Protein-protein Interactions by Formaldehyde Cross-linking in Living Cells
<ol>
	<li>To begin prepare growth media, stock solutions and buffer P1
	<ol>
		<li>Prepare 500 ml medium: The medium should provide conditions that support the interaction between membrane bait protein and prey proteins. In addition, one experiment should be performed under conditions where n...

<ol>
	<li>Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+transmembrane+protein+topology+with+a+hidden+markov+model:+application+to+complete+genomes.">Predicting transmembrane protein topology with a hidden markov model: application to complete genomes.</a> J. Mol. Biol. 305, 567-580 (2001).</li><li>Bakheet, T. M., Doig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+identification+of+human+protein+drug+targets.">Properties and identification of human protein drug targets.</a> Bioinformatics. 25, 451-457 (2009).</li><li>Yildirim, M. A., Goh, K. I., Cusick, M. E., Barabasi, A. L., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-target+network.">Drug-target network.</a> Nat. Biotechnol. 25, (2007).</li><li>Daley, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assembly+of+membrane+proteins+into+complexes.">The assembly of membrane proteins into complexes.</a> Curr. Opin. Struct. Biol. 18, 420-424 (2008).</li><li>Jung, K., Fried, L., Behr, S., Heermann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histidine+kinases+and+response+regulators+in+networks.">Histidine kinases and response regulators in networks.</a> Curr. Opin. Microbiol. 15, 118-124 (2012).</li><li>M&#252;ller, V. S., Jungblut, P. R., Meyer, T. F., Hunke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-SPINE:+An+improved+method+to+identify+protein-protein+interaction+partners+of+membrane+proteins+in+vivo.">Membrane-SPINE: An improved method to identify protein-protein interaction partners of membrane proteins in vivo.</a> Proteomics. 11, 2124-2128 (2011).</li><li>Herzberg, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPINE:+A+method+for+the+rapid+detection+and+analysis+of+protein-protein+interactions+in+vivo.">SPINE: A method for the rapid detection and analysis of protein-protein interactions in vivo.</a> Proteomics. 7, 4032-4035 (2007).</li><li>Sutherland, B. W., Toews, J., Kast, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+formaldehyde+cross-linking+and+mass+spectrometry+in+the+study+of+protein-protein+interactions.">Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions.</a> J. Mass Spectrom. 43, 699-715 (2008).</li><li>Hernandez, P., M&#252;ller, M., Appel, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+protein+identification+by+tandem+mass+spectrometry:+Issues+and+strategies.">Automated protein identification by tandem mass spectrometry: Issues and strategies.</a> Mass Spectrom. Rev. 25, 235-254 (2006).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Hunke, S., Keller, R., M&#252;ller, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+by+the+Cpx-envelope+stress+system.">Signal integration by the Cpx-envelope stress system.</a> FEMS Microbiol. Lett. 326, 12-22 (2012).</li><li>Weber, R. F., Silverman, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cpx+proteins+of+Escherichia+coli+K12+:+Structure+of+the+CpxA+polypeptide+as+an+inner+membrane+component.">The Cpx proteins of Escherichia coli K12 : Structure of the CpxA polypeptide as an inner membrane component.</a> J. Mol. Biol. 203, 467-478 (1988).</li></ol>

Membrane SPINE analysis is a biochemical approach that enables one to confirm and to identify to this point unknown interaction partners of membrane proteins. Membrane SPINE combines in vivo cross-linking by formaldehyde with purification of a Strep-tagged membrane bait protein. The combination with immunoblotting facilitates the confirmation of predicted interaction partners (Figure 2). Additionally, the combination with MS analysis permits the identification of unknown interaction partners (<s...

To understand the function of a protein it is essential to know its interaction partners. Several classical techniques are available for the identification of interaction partners of soluble proteins. However, these techniques are not easily transferable to membrane proteins due to their hydrophobic nature4. To overcome this limitation, we have developed the Membrane Strep-protein interaction experiment (Membrane-SPINE)6. It is based on the SPINE method, which was only suitable for soluble proteins7.
Membrane-SPINE benefits from two advantages of the cross-linking agent formaldehyde: first, formaldehyde can easily penetrate membranes and therefore generates a precise snapshot of the interactome of a living cell8. Second, formaldehyde cross-links can be reversed by boiling9. Here, these two advantages are used to identify not only permanent but also transient PPIs of membrane proteins6.
In brief, a Strep-tag is fused to the C-terminus of the integral membrane bait protein. Cells expressing the membrane bait protein are incubated with formaldehyde which cross-links prey proteins to the membrane bait protein (Figure 1). Modifications of prey proteins are not needed. Next, the membrane fraction is prepared. Therefore, membrane proteins are solubilized by detergent treatment and bait proteins are co-purified with its prey proteins using affinity purification. Subsequently, the cross-link is reversed by boiling, and the bait and its co-eluted prey proteins are separated by SDS-PAGE. Finally, prey proteins can be identified by immunoblot analysis or mass spectrometry.

<table border="0" cellpadding="0" cellspacing="0" width="878">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">37% Formaldehyde</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">4979.1</td>
			<td style="width:271px;">Should not be older than one year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DNaseI</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:137px;">DN25</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BCA protein assay kit</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">23225</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Micromagnetic rod</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">0955.2</td>
			<td style="width:271px;">5 mm in length, 2 mm in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Triton X-100</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">6683.1</td>
			<td style="width:271px;">standard detergent for solubilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">n-Dodecyl-&#946;-maltoside</td>
			<td style="width:128px;">Glycon</td>
			<td style="width:137px;">D97002-C</td>
			<td style="width:271px;">the best detergent to solubilze CpxA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">1 ml Strep-Tactin superflow gravity flow column</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1207-050</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Strep-tag protein purification buffer set</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1002-001</td>
			<td style="width:271px;">Contains buffer W and buffer E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Amicon Ultra-4 centrifugal filter</td>
			<td style="width:128px;">Millipore</td>
			<td style="width:137px;">UFC803024</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">SuperSignal West Pico Chemiluminescent substrate</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">34080</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SilverQuest Silverstaining kit</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:137px;">LC6070</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FireSilver Staining Kit</td>
			<td style="width:128px;">Proteome Factory</td>
			<td style="width:137px;">P-S-2001</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Ultracentrifuge</td>
			<td style="width:128px;"></td>
			<td style="width:137px;"></td>
			<td style="width:271px;"></td>
		</tr>
	</tbody>
</table>

membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane SPINE analysis allows the co-purification of membrane proteins and transiently interacting protein partners. The co-purification is achieved by using the cross-linking agent formaldehyde. Two parameters are critical to prevent unspecific cross-links: the formaldehyde concentration and the cross-linking time. The sufficient, but not excessive use of formaldehyde can easily be controlled by immunoblotting. Formaldehyde cross-linked protein complexes can be separated by boiling but not by SDS treatment. Hence, they...

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Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

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13.6K Views.  Universität Osnabrück. A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.