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Abstract
In-gel digestion has been used as a standard method for the preparation of protein samples for mass spectrometry analysis for over 25 years. Traditional in gel-digestion procedures require extensive sample handling, are prone to contamination and not compatible with high-throughput sample preparation. To address these shortcomings, we have modified the conventional in-gel digestion procedure for high-throughput proteomics studies. The modified method, termed “High Throughput in Gel digestion” (HiT-Gel), is based on a 96-well plate format which results in a drastic reduction in labour intensity and sample handling. Direct comparison revealed that HiT-Gel reduces technical variation and significantly decreases sample contamination over the conventional in-gel digestion method. HiT-Gel also produced superior results when a single protein band was excised from a gel and processed by in-gel digestion. Moreover, we applied Hit-Gel for a mass spectrometry analysis of Arabidopsis thaliana protein complexes separated by native PAGE in 24 fractions and four biological replicates. We show that the high throughput capacity of HiT-Gel facilitates large scale studies with high sample replication or detailed fractionation. Our method can easily be implemented as it does not require specialised laboratory equipment.
Highlights
In-gel digestion is a standard method to prepare proteins obtained from biological samples for mass-spectrometry (MS)-based analysis
In-gel digestion remains essential in workflows to identify and quantify by mass spectrometry the components of protein complexes fractionated by native PAGE
The results show that High Throughput in Gel digestion” (HiT-Gel) is a major advancement with regards to reduction of workload and sample contamination and increased reproducibility
Summary
In-gel digestion is a standard method to prepare proteins obtained from biological samples for mass-spectrometry (MS)-based analysis. As gel fractions are kept intact in the HiT-Gel method, the risk of picking up and discarding small gel pieces with a pipette tip during the repeated exchange of solutions is reduced in comparison to the conventional method Another important alteration to the conventional method is that the gel pieces are no longer transferred to microfuge tubes but to 96-well plates, where all sample preparation steps are performed with the aid of multichannel pipettes. The results show that HiT-Gel is a major advancement with regards to reduction of workload and sample contamination and increased reproducibility It facilitates high-throughput sample preparation, which could especially be of interest for quantitative, gel-based studies of native protein complexes with detailed fractionation and sample replication
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