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Related Experiment Video

Updated: Jul 23, 2025

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology
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Analyzing Protein Interactions by MAC-Tag Approaches.

Xiaonan Liu1,2, Kari Salokas1, Salla Keskitalo1

  • 1Institute of Biotechnology, HiLIFE Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.

Methods in Molecular Biology (Clifton, N.J.)
|July 14, 2023
PubMed
Summary

We enhanced the Multiple Approaches Combined (MAC)-tag workflow for proteomics by integrating newer proximity-dependent labeling (PL) tags, BioID2 and UltraID. This improves protein interaction analysis using mass spectrometry (MS).

Keywords:
Affinity purificationInteractomicsMAC-tagMAC3-tagMass spectrometryProtein interactionsProteomicsProximity labeling

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Area of Science:

  • Molecular Biology
  • Proteomics
  • Biochemistry

Background:

  • Affinity purification (AP) and proximity-dependent labeling (PL) coupled with mass spectrometry (MS) are key for mapping protein interactions.
  • The original Multiple Approaches Combined (MAC)-tag workflow facilitates both AP and BioID analysis from a single construct.
  • Newer PL tags like BioID2 and UltraID offer smaller size and enhanced labeling efficiency compared to the original BioID system.

Purpose of the Study:

  • To develop improved MAC-tag systems (MAC2-tag and MAC3-tag) by incorporating BioID2 and UltraID, respectively.
  • To overcome limitations of the original MAC-tag system and expand its applicability in proteomics research.
  • To provide a detailed protocol for generating cell lines, sample preparation, and MS analysis for the enhanced MAC-tag workflows.

Main Methods:

  • Development of MAC2-tag and MAC3-tag systems by fusing proteins of interest (POI) to BioID2 and UltraID, respectively.
  • Generation of stable cell lines expressing the MAC/MAC2/MAC3-tagged POI.
  • Standardized protocols for AP and PL protein purification followed by MS identification.

Main Results:

  • Successful integration of BioID2 and UltraID into the MAC-tag workflow, creating MAC2-tag and MAC3-tag systems.
  • Demonstrated feasibility of using the new systems for AP and PL analyses with streamlined MS procedures.
  • The enhanced systems are expected to improve the efficiency and scope of protein interaction studies.

Conclusions:

  • The MAC2-tag and MAC3-tag workflows represent significant advancements over the original MAC-tag system.
  • These updated methods provide researchers with more versatile and efficient tools for comprehensive proteomic analyses.
  • The detailed protocol facilitates the adoption and application of these enhanced techniques in various biological studies.