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

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

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell

DISEASE(S): Acute Leukemia

SUBMITTER: Ching-Seng Ang  

LAB HEAD: Nicholas A. Williamson

PROVIDER: PXD028965 | Pride | 2023-02-21

REPOSITORIES: Pride