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Updated: Oct 4, 2025
Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
High-throughput split-protein profiling by combining transposon mutagenesis and regulated protein-protein
Kai Zhou1, Thomas Litfin2, Md Solayman2
1Institute for Glycomics and School of Information and Communication Technology, Griffith University, Parklands Dr Southport, QLD 4222, Australia; Institute for Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen 518055, China.
This study introduces a high-throughput technique (HiTS) for rapidly identifying self- and assisted-complementary protein split sites. This method accelerates the discovery of protein variants crucial for probing biological interactions.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Protein splitting can yield self- or assisted-complementary fragments, essential for studying biological interactions.
- Identifying these fragments is challenging due to manual, labor-intensive gene optimization.
Purpose of the Study:
- To develop a high-throughput method for fast identification of self- and assisted-complementary protein split positions.
- To enable efficient discovery of protein variants for biological interaction studies.
Main Methods:
- Developed high-throughput split-protein profiling (HiTS) using transposon mutagenesis, a rapamycin-regulated FRB-FKBP interaction pair, and deep sequencing.
- Applied HiTS to profile three antibiotic-resistant genes: fosA3, ermB, and catI.
- Confirmed HiTS-identified split positions through low-throughput testing.
Main Results:
- Successfully identified self- and assisted-complementary fragments in antibiotic-resistant genes using the HiTS technique.
- Demonstrated the speed and efficiency of HiTS compared to traditional methods.
- Validated the discovered split positions through subsequent low-throughput experiments.
Conclusions:
- The HiTS technique offers a rapid and efficient alternative for discovering proteins with suitable self- and assisted-complementary split positions.
- This method can be combined with various readouts like fluorescence or gene editing for broader applications.
- Facilitates the study of protein interactions and the development of novel biological tools.
