Ruprecht-Karls-Universität Heidelberg

Contact us:
Zentrum fr Molekulare Biologie der Universitt Heidelberg (ZMBH)
Im Neuenheimer Feld 282
Room 401
69120 Heidelberg, Germany
Tel.: +49-6221-54 6813
Fax: +49-6221-54 5894


Co-IP or co-affinity purification MS

For the bulk identification of protein interaction partners, we commonly apply co-immunoprecipitation/co-affinity purification together with quantitative mass spectrometry (Figure 1). For this, the protein of interest is precipitated from cells together with its interacting proteins, which are subsequently identified by mass spectrometry. Because such precipitates will also contain contaminants (e.g. unspecific binders to affinity beads, IgG beads, antibody,...), a control sample not containing the bait is needed. In the end, true interactors can be distuinguished from contaminants by looking at their respective abundances in both samples. While contaminants are equally present in both samples, specific interactors are enriched in the bait sample. For determining the proteins abundances different quantification strategies are available, both label-free (spectral count, MaxQuant-LFQ) and label-based (SILAC, DML) (see Quantitative Proteomics).

Identifying interaction partners by IP
                        and spectral counting
Figure 1 - Schematic of interaction partner screening by co-affinity purification plus spectral count quantitative MS. In this example, an affinity tag (e.g.: YFP, StrepTag, FLAG, HA,...) is attached to the bait protein for co-enrichment. Alternatively, direct precipication by specific antibodies is also possible.

Cross-linking MS

For elucidating protein-protein interaction surfaces/sites we utilize the cross-link MS method, developed by the Aebersold lab1,2, which relies on the usage of isotopically labeled cross-linkers. The figures below describe the general worklfow of such an experiment.

crosslinking01 crosslinking ms02
Figure 2 - Workflow of Cross-Linking MS experiment

1. Leitner, A., et al. (2012) Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography, Molecular and Cellular Proteomics, 11 (3), (p. M111.014126) - Pubmed
2. Leitner, A., et al. (2014) Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC-MS/MS and the xQuest/xProphet software pipeline, Nature protocols, 9 (1), (doi:10.1038/nprot.2013.168)