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Related Experiment Video

Updated: Nov 19, 2025

Mapping Dysfunctional Protein-Protein Interactions in Disease
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Fully Integrated and Multiplexed Sample Preparation Technology for Sensitive Interactome Profiling.

Yiheng Mao1,2, Peizhong Chen2,3, Mi Ke2

  • 1School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China.

Analytical Chemistry
|February 1, 2021
PubMed
Summary
This summary is machine-generated.

We developed FISAP, a spintip-based affinity purification coupled to mass spectrometry (AP-MS) technology. This method enables sensitive interactome profiling using significantly less protein lysate, improving quantification and reducing background noise.

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Area of Science:

  • Proteomics
  • Biochemistry
  • Molecular Biology

Background:

  • Affinity purification coupled to mass spectrometry (AP-MS) is crucial for mapping protein interaction networks.
  • Traditional AP-MS methods require substantial protein amounts (e.g., 100 μg), leading to sample loss and reduced sensitivity.
  • There is a need for more sensitive and efficient AP-MS techniques, especially when starting material is limited.

Purpose of the Study:

  • To develop an integrated, spintip-based AP-MS technology (FISAP) for sensitive and multiplexed interactome profiling.
  • To reduce sample input requirements and improve quantification performance compared to conventional methods.
  • To demonstrate the broad applicability of FISAP for various AP-MS strategies.

Main Methods:

  • Developed a fully integrated spintip device (FISAP) for streamlined sample preparation within a single tip.
  • Integrated affinity purification (AP), reduction, alkylation, tryptic digestion, tandem mass tag (TMT) labeling, and desalting steps.
  • Utilized switchable functionalization of a C18 matrix for efficient sample processing.

Main Results:

  • FISAP successfully mapped the pTyr-dependent interactome of CD28 using only 50 μg of protein lysate, showing comparable identification but superior quantification and lower background than tube-based methods.
  • An optimized on-column TMT labeling protocol reduced reagent usage by 20-fold, enabling interactome profiling with as little as 1 μg of lysate.
  • Applied FISAP to epitope tag-based AP-MS for profiling the ILK/PINCH/Parvin complex with 100-fold less starting material than previously reported.

Conclusions:

  • FISAP is an easy-to-use, sensitive, and quantitative technology for interactome profiling, particularly advantageous when protein or affinity reagent amounts are limiting.
  • The technology significantly reduces sample input and reagent consumption, making it ideal for biomedical research and chemical biology.
  • FISAP offers a robust platform for advancing the study of protein complexes and biological pathways.