An Optimized Protocol for TurboID-Based Proximity Labeling and Sample Preparation for Mass Spectrometry in Arabidopsis thaliana
- 1Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, China
Abstract
Plant proximity labeling proteomics enables the identification of transient and weak intracellular protein interactions that are undetectable via traditional biochemical assays. Conventional enrichment pipelines suffer severe quantitative bias caused by urea-derived chemical artifacts and high mass spectrometry background signals from sample- and carrier-derived impurities. This protocol describes a complete standardized workflow for biotinylated protein extraction, enrichment, and LC-MS/MS sample preparation optimized for Arabidopsis seedlings. The procedure integrates controlled urea dilution and pre-desalting to suppress non-enzymatic protein modifications, introduces acetylation blocking of streptavidin magnetic beads to mitigate streptavidin degradation during on-bead digestion, and applies two-step on-bead trypsin digestion to improve peptide coverage. Multi-point sample retention and dual protein quantification are implemented throughout all experimental stages to ensure full-process quality control. Subsequent data processing pipelines using Spectronaut for data-independent acquisition (DIA) data and MaxQuant for data-dependent acquisition (DDA) data are also detailed for consistent proteome quantification. This workflow delivers higher protein recovery and better signal-to-noise ratios than standard protocols while offering flexible adaptation for various crop proximity labeling and affinity enrichment proteomic research.
Key features
• Complete standardized preprocessing pipeline specially optimized for Arabidopsis seedlings undergoing TurboID-based proximity labeling proteomics.
• Urea dilution and pre-desalting to suppress artificial protein carbamylation.
• Magnetic bead acetylation blocking to mitigate streptavidin degradation during on-bead digestion, coupled with two-step tryptic digestion for higher peptide coverage.
• Full-process multi-point quality control, compatible with DIA (Spectronaut) and DDA (MaxQuant) mass spectrometry analysis.
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BibTeX
@article{zhang2026optimized,
title = {{An Optimized Protocol for TurboID-Based Proximity Labeling and Sample Preparation for Mass Spectrometry in Arabidopsis thaliana}},
author = {Li-Dan Zhang and Yu-Qi Ye and Dan-Ting Zhuo and Qiu-Ping Yan and Ya-Ling Ye and Hongbo Li and Caiji Gao and Lei Feng},
journal = {Bio-protocol},
year = {2026},
month = oct,
doi = {10.21769/bioprotoc.5852},
url = {https://doi.org/10.21769/bioprotoc.5852},
note = {CC BY-NC 4.0},
}
RIS
TY - JOUR
TI - An Optimized Protocol for TurboID-Based Proximity Labeling and Sample Preparation for Mass Spectrometry in Arabidopsis thaliana
AU - Li-Dan Zhang
AU - Yu-Qi Ye
AU - Dan-Ting Zhuo
AU - Qiu-Ping Yan
AU - Ya-Ling Ye
AU - Hongbo Li
AU - Caiji Gao
AU - Lei Feng
PY - 2026
DA - 2026/10/20
JO - Bio-protocol
DO - 10.21769/bioprotoc.5852
UR - https://doi.org/10.21769/bioprotoc.5852
AB - Plant proximity labeling proteomics enables the identification of transient and weak intracellular protein interactions that are undetectable via traditional biochemical assays. Conventional enrichment pipelines suffer severe quantitative bias caused by urea-derived chemical artifacts and high mass spectrometry background signals from sample- and carrier-derived impurities. This protocol describes a complete standardized workflow for biotinylated protein extraction, enrichment, and LC-MS/MS sample preparation optimized for Arabidopsis seedlings. The procedure integrates controlled urea dilution and pre-desalting to suppress non-enzymatic protein modifications, introduces acetylation blocking of streptavidin magnetic beads to mitigate streptavidin degradation during on-bead digestion, and applies two-step on-bead trypsin digestion to improve peptide coverage. Multi-point sample retention and dual protein quantification are implemented throughout all experimental stages to ensure full-process quality control. Subsequent data processing pipelines using Spectronaut for data-independent acquisition (DIA) data and MaxQuant for data-dependent acquisition (DDA) data are also detailed for consistent proteome quantification. This workflow delivers higher protein recovery and better signal-to-noise ratios than standard protocols while offering flexible adaptation for various crop proximity labeling and affinity enrichment proteomic research. Key features • Complete standardized preprocessing pipeline specially optimized for Arabidopsis seedlings undergoing TurboID-based proximity labeling proteomics. • Urea dilution and pre-desalting to suppress artificial protein carbamylation. • Magnetic bead acetylation blocking to mitigate streptavidin degradation during on-bead digestion, coupled with two-step tryptic digestion for higher peptide coverage. • Full-process multi-point quality control, compatible with DIA (Spectronaut) and DDA (MaxQuant) mass spectrometry analysis.
SN - 2331-8325
ER -