Sample preparation for structural mass spectrometry via polyacrylamide gel electrophoresis.

Mass spectrometry is an analytical technique that can detect protein molecules with high sensitivity. Its use is not limited to the mere identification of protein components in biological samples, but is recently being utilized for large-scale analysis of protein structures in vivo as well. Top-down mass spectrometry with an ultra-high resolution mass spectrometer, for example, ionizes proteins in their intact state and allows rapid analysis of their chemical structure, which is used to determine proteoform profiles. Furthermore, cross-linking mass spectrometry, which analyzes enzyme-digested fragments of chemically cross-linked protein complexes, allows acquisition of conformational information on protein complexes in multimolecular crowding environments. In the analysis workflow of structural mass spectrometry, prior fractionation of crude biological samples is an effective way to obtain more detailed structural information. Polyacrylamide gel electrophoresis (PAGE), known as a simple and reproducible means of protein separation in biochemistry, is one example of an excellent high-resolution sample prefractionation tool for structural mass spectrometry. This chapter describes elemental technologies for PAGE-based sample prefractionation including Passively Eluting Proteins from Polyacrylamide gels as Intact species for Mass Spectrometry (PEPPI-MS), a highly efficient method for intact in-gel protein recovery, and Anion-Exchange disk-assisted Sequential sample Preparation (AnExSP), a rapid enzymatic digestion method using a solid-phase extraction microspin column for gel-recovered proteins, in addition to presenting detailed experimental protocols and examples of their use for structural mass spectrometry.