Getting more out of FLAG-Tag co-immunoprecipitation mass spectrometry experiments using FAIMS

Co-immunoprecipitation of proteins coupled to mass spectrometry is critical for the understanding of 24 protein interaction networks. In instances where a suitable antibody is not available, it is common to graft 25 synthetic tags onto target protein sequences and allowing the use of commercially available antibodies 26 for affinity purification. A common approach is through FLAG-Tag co-immunoprecipitation. To allow the 27 selective elution of protein complexes, competitive displacement using a large molar excess of the tag 28 peptides is often carried out. Yet, this creates downstream challenges for the mass spectrometry analysis 29 due to the presence of large quantities of these peptides. Here, we demonstrate that Field Asymmetric 30 Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device prior to mass spectrometry analysis 31 can be applied to FLAG-Tag co-immunoprecipitation experiment to increase the depth of protein 32 coverage. By excluding these abundant tag peptides, we were able to observe deeper coverage of 33 interacting proteins and as a result, deeper biological insights, without the need for additional sample 34 handling or altering sample preparation protocols.