Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared

Claudia Maria do Nascimento Moreira, Cristina D. Kelemen, Samson O. Obado, Farnaz Zahedifard, Ning Zhang, Fabiola B. Holetz, Laura Gauglitz, Bruno Dallagiovanna, Mark C. Field, Susanne Kramer (Lead / Corresponding author), Martin Zoltner (Lead / Corresponding author)

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The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: Affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, while in BioID genetic fusion of biotin ligase facilitates proximity biotinylation and labelled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A) binding proteins PABP1 and PABP2, mRNA export receptor MEX67 and the nucleoporin NUP158. With BioID, we found the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false-positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, while the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within outer rings of the NPC that were by contrast detected with pulldown, but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

Original languageEnglish
Article number102726
Number of pages15
JournalJournal of Biological Chemistry
Issue number1
Early online date18 Nov 2022
Publication statusPublished - Jan 2023


  • affinity capture
  • proteome
  • cryomilling
  • BioID
  • interactome


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