GeLCMS for In-Depth Protein Characterization and Advanced Analysis of Proteomes
In recent years the array of mass spectrometry (MS) applications to address questions in molecular and cellular biology has greatly expanded and continues to grow. Modern mass spectrometers allow for identification, characterization, as well as quantification of protein compositions and their modifications in complex biological samples. Prior to MS analysis any biological sample needs to be properly prepared for the experiment. Here we present a protocol that combines pre-separation of proteins by 1D gel electrophoresis followed by analysis of in situ digested protein products by tandem mass spectrometry (MS/MS). All steps of the sample preparation are explained in detail, and the procedure is compatible with downstream analysis on any mass spectrometer available. With minor adjustments the protocol can be used with 2D gels as well. The protocol provided can be applied to analyze specific proteins of particular interest as well as entire proteomes. If SILAC-labeled protein samples are mixed prior to gel separation, the protein content of the sample can furthermore be accurately quantified.
Key wordsIn-gel digest mass spectrometry proteomics LTQ-Orbitrap Velos protein identification post-translational modifications LC-MS/MS GeLCMS
The authors thank all members of the Department for Proteomics at the Novo Nordisk Foundation Center for Protein Research for helpful scientific discussions and in particular Brian Tate Weinert for feedback on the chapter. This work was in part supported by the Danish National Research Foundation Centre for Cardiac Arrhythmia. The NNF Center for Protein Research is supported by a generous donation from the Novo Nordisk Foundation.
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