Abstract
The elucidation of protein–protein interaction networks is a crucial task in the postgenomic era. In this protocol, we describe our approach to discover protein–protein interactions using the surface plasmon resonance technique coupled to mass spectrometry (MS). A peptide or a protein is immobilized on a sensor chip and then exposed to brain extracts injected through the surface of the chip by a microfluidic system. The interactions between the immobilized ligand and the extracts can be monitored in real time. Proteins interacting with the peptide/protein are recovered, trypsinated and identified using MS. The data obtained are searched against a sequence database using the Mascot 2.1 software. Control experiments using blank sensor chips and/or randomized peptides are carried out to exclude nonspecific interactors. The protocol can be carried out in <3 days. Other methods, such as yeast two-hybrid systems or pull-down approaches followed by MS, are widely used to screen protein–protein interactions. However, as the yeast two-hybrid system requires protein interactions in the nucleus of yeast, proteins that are abundant in other compartments may not be detected. Pull-down approaches based on immunoprecipitation can be used to study endogenous proteins but they require specific antibodies. The protocol presented here does not require the specific labeling or modification of proteins.
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Acknowledgements
This work was supported by the Swedish Research Council (Vetenskapsrådet, VR), the French Foundation for Medical Research (Fondation pour la Recherche Médicale FRM) and the Torgny and Ragnar Söderberg's Foundation (Söderberg Stiftelse).
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Supplementary Figure 1 Program to immobilize a peptide on a carboxymethylated sensor chip.
This program consists of a sequence of commands controlling the immobilization experiment. A translation of these commands can be found in the procedure. Exclamation points allow adding some comments which are not included in the operation. (PPT 128 kb)
Supplementary Figure 2 Program to bind and recover proteins against a peptide on a carboxymethylated sensor chip.
This program consists of a sequence of commands controlling the recovery experiments in the instrument. A translation of these commands can be found in the procedure. Exclamation points allow adding some comments which are not included in the operation. (PPT 130 kb)
Supplementary Figure 3 Program to bind and recover proteins against a peptide on a carboxymethylated chip in the surface prep unit.
This program consists of a sequence of commands controlling the recovery experiments in the surface prep unit. A translation of these commands can be found in the procedure. Exclamation points allow adding some comments which are not included in the operation. (PPT 130 kb)
Supplementary Figure 4 Mass spectra assigned to the peptide ASFTTFTVTK derived from Caveolin-1 (P41350).
The spectra contain the parent mass, charge and observed fragmentation pattern in the form of peak list. Monoisotopic mass of neutral peptide Mr (calc): 1101.57. Ions Score: 64; Expect: 1.7e-005. Matches (Bold Red): 12/86 fragment ions using 24 most intense peaks (modified and reproduced with permission from 5). All animal experiments are performed according to the local ethical committee at the Karolinska Institute (Application N282/06) (PPT 91 kb)
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Madeira, A., Öhman, E., Nilsson, A. et al. Coupling surface plasmon resonance to mass spectrometry to discover novel protein–protein interactions. Nat Protoc 4, 1023–1037 (2009). https://doi.org/10.1038/nprot.2009.84
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DOI: https://doi.org/10.1038/nprot.2009.84
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