STAGE-diging: A novel in-gel digestion processing for proteomics samples.

UNLABELLED: Proteomics based on high-resolution mass spectrometry has become a powerful tool for the analysis of protein abundance, modifications and interactions. New generation mass spectrometers and UPLC are able to cover approximately an entire cell proteome in one run, but sample preparation, in terms of time and sample recovery is still a critical step. Here we present a modification of the in-gel digestion method, called STAGE-diging. This approach was compared with the well-established procedures for sample preparation, both on high and low complexity samples, on quantitative SILAC-based experiments and on two different mass spectrometers. The results show that STAGE-diging reduces sample handling, decreases the analysis time and improves protein identification and quantification. Moreover, shorter instrument time allows performing multiple replicates that produce wider proteome coverage and more accurate quantitation. SIGNIFICANCE: In our work we detailed the set-up of a novel in-gel digestion processing for proteomics samples, called STAGE-diging. This new method can be applied to samples of different complexity both for qualitative and quantitative proteomics studies. We proved that STAGE-diging streamlines sample preparation as it is easy to use, reduces sample handling and improves protein identification and quantification all with a decreased analysis time. All these benefits make this new method appealing for laboratories handling a large number of samples, where time and reproducibility play a substantial role.