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Updated: Sep 16, 2025

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

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Published on: November 15, 2017

Optimizing Proximity Proteomics on the EvoSep-timsTOF LC-MS System.

Julia Kitaygorodsky^1,2, Brendon Seale¹, Vesal Kasmaeifar^1,2

¹Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health, Toronto, Ontario, Canada.

|July 11, 2025

Summary

This summary is machine-generated.

Optimizing proximity-dependent biotinylation (BioID) with EvoSep-timsTOF mass spectrometry increases throughput 15-fold and identifies more proteins. This new workflow minimizes peptide carryover, eliminating wash steps and enabling 60 samples daily.

Keywords:

BioID EvoSep Significance Analysis of INTeractome cytoskeleton interactions protein–protein interactions proximity‐dependent biotinylation spectral counts timsTOF

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Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

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Published on: April 1, 2017

Area of Science:

Proteomics
Biochemistry
Cell Biology

Background:

Proximity-dependent biotinylation (BioID) identifies protein interactors by labeling proximal proteins with biotin.
Peptide carryover in mass spectrometry (MS) limits BioID throughput due to necessary wash cycles.
Existing BioID workflows require lengthy intersample washes, reducing sample processing efficiency.

Purpose of the Study:

To optimize BioID sample acquisition using an EvoSep LC system coupled to a timsTOF mass spectrometer.
To increase throughput and sensitivity while reducing peptide carryover in BioID-MS workflows.
To systematically evaluate the EvoSep-timsTOF system for BioID and compare it to established methods.

Main Methods:

Implemented an EvoSep LC system with a timsTOF mass spectrometer for BioID sample analysis.
Optimized sample loading and gradient settings for the EvoSep-timsTOF platform.
Compared protein identification and carryover levels against a previously standardized BioID workflow.

Main Results:

Achieved an approximately 15-fold increase in throughput, processing 60 samples per day.
Identified nearly double the number of proteins compared to the previous workflow with enhanced sensitivity.
Demonstrated extremely limited peptide carryover, even without intersample washing, allowing for elimination of wash cycles.

Conclusions:

The EvoSep-timsTOF system significantly enhances BioID throughput and sensitivity.
Eliminating intersample washing is feasible without compromising the identification of proximal interactors.
Optimized BioID workflows using advanced instrumentation improve the efficiency and scope of proteomic studies.