Abstract
Co-immunoprecipitation of proteins coupled to mass spectrometry has transformed modern biology’s understanding of protein interaction networks. These approaches exploit the selective isolation of tagged proteins by affinity enrichment / purification to identify protein binding partners at scale and in an unbiased manner. In instances where a suitable antibody is not be available it is common to graft synthetic tags such as FLAG or His Tags onto target protein sequences allowing the use of commercially available and validated antibodies for affinity purification. To allow the selective elution of protein complexes competitive displacement using a large molar excess of the tag peptide is widely used. Yet, this creates downstream challenges for the mass spectrometry analysis due to the presence of large quantities of a contaminating peptide. Here, we demonstrate that Field Asymmetric Ion Mobility Spectrometry (FAIMS), a gas phase ion separation device can be applied to FLAG-Tag and His-Tag pull down assay to increase the depth of protein coverage in these experiments. By excluding tag peptides based on their ion mobility profiles we demonstrate that single compensation voltage, or stepped compensation voltages strategies can significantly increase the coverage of total proteins by up to 2.5-fold and unique proteins by up to 15-fold versus experiments that do not use FAIMS. Combined these results highlight FAIMS is able to improve proteome depth by excluding interfering peptides without the need for additional sample handling or altering sample preparation protocols.
Significance Statement We have shown that application of FAIMS separation in the gas phase can significantly increase the proteome coverage of Flag or His tagged co-immunoprecipitation mass spectrometry experiments. We demonstrated this in a classical FLAG tagged and His Tag assay and have increased the proteome coverage by up to 15-fold versus one without FAIMS. This allows us to conduct such experiment without additional sample handling, fractionation, machine run time or modifying the sample preparation protocol.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Author Contributions: CSA and NAW designed the research, CSA developed methodology, CSA, MGL, SN, SV performed experiments, all authors analyzed data and wrote the paper.