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Non-specific binding possibilities should always be assessed for each set of experimental parameters by performing a control IP exactly the same way as for the IP of interest, with a control antibody or beads alone. Proteins identified in the control IP can be considered as putative contaminants. However, to be reliable, the comparison between the control IP and the IP of interest needs to be quantitative.

The combination of quantitative MS and differential labelling of proteins with heavy isotopes, especially stable isotope labelling with amino-acids in cell culture (SILAC) (Ong, Blagoev et al. 2002) allows for direct quantitative comparison between the relative levels of each protein present in the control and experimental samples, through the inclusion of an internal control in the experiment. This approach eliminates the variations between runs and is therefore very powerful to distinguish between specific and non-specific binding proteins in a co-IP experiment. A triple labelling SILAC strategy can also be used to study the dynamics of protein complexes between different conditions (Boulon, Ahmad et al. 2010).

A standard workflow for triple labelling SILAC-based pull-down experiments in mammalian cells is summarized below. Basically, three separate growth conditions are used to label cells with different isotopes that can be resolved and quantitated by MS. An internal control is provided by cells grown in light (L), i.e., unlabeled (R0K0: 12C, 14N, 1H), medium, whereas the IP of interest is performed with extracts from cells that are grown either with medium (M) ([13C6]arginine (R6) and 4,4,5,5-D4-lysine (K4)) or with heavy (H) ([13C6, 15N4]arginine (R10) and [13C6, 15N2]lysine (K8)) isotope-labeled arginine and lysine amino-acids. The cells grown in medium and heavy media are used to compare changes in specific protein interaction partners between (i) control conditions and treatment with chemical inhibitors/stress etc, (ii) wild-type and mutant forms of the bait protein, or (iii) two different isoforms of the same protein.

A typical experimental procedure for both GFP and endogenous co-IP triple SILAC experiment is presented below. In brief, cell lysates are prepared from each of the L, M and H cell cultures, and the bait protein and associated interaction partners are immunoaffinity-purified. Eluted proteins are then in-gel digested with trypsin (or other proteases), and peptides are analysed by LC-MS/MS and quantified using MaxQuant (Cox and Mann 2008; Cox, Matic et al. 2009).

Each identified peptide shows a typical MS spectrum with three main peaks that correspond to the light, medium and heavy isotopic forms. The relative abundance of each distinct peak area can be efficiently quantified by MaxQuant, which indicates three median ratios (M/L, H/L, and H/M) for each peptide as well as the median value for all peptides quantitated for a specific protein. In this strategy, both the bait and its protein interaction partners are expected to have a higher M/L and/or H/L ratio than non-specific contaminants, which should have M/L and H/L ratios close to 1. Theoretically,

• Specific interaction partners: ratio > 1.4
• Experimental contaminants: 0.6
• Environmental contaminants (e.g., keratins): ratio <0.6

In addition, while M/L and H/L ratios allow for the discrimination between contaminants and genuine interaction partners, H/M ratios indicate changes in protein complexes under specific biological conditions.