Hit-Gel: Streamlining in-gel protein digestion for high-throughput proteomics experiments.

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 formatwhich 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 thalianaprotein complexesseparated by native PAGE in 24 fractions and four biological replicates. We show that the high throughput capacity ofHiT-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.