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Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling
Catrin S. Mueller, Wolfgang Bildl, Alexander Haupt, Lars Ellenrieder, Thomas Becker, Carola Hunte, Bernd Fakler and Uwe Schulte
Molecular & Cellular Proteomics (2016), 15, 669-681
In this study the authors present a new high resolution workflow for characterization of native protein complexes. Using blue native (BN) gel electrophoresis combined with LC-MS/MS, it enables large scale identification of complexes and their subunits. This BN-MS workflow provides several advantages over previous approaches - it uses nearly the full resolving power of the BN-PAGE separation, it provides detailed abundance-mass profiles over a wide molecular size range, and it provides more comprehensive resolution of the subunit composition.
Sample preparation is performed by gel slicing with a cryo-microtome. For the analysis of a rat brain mitochondrial membrane preparation, it provided a total of 230 slices, each covering 0.3 mm of the BN gel. These gel slice samples were subsequently digested and analyzed by LC-MS/MS, and the data were evaluated using label-free quantification of proteins. In the entire set of cryo-slices, these analyses identified over 1,200 proteins, 774 of which were mitochondrial proteins.
Besides the higher resolution, the method was able to identify and quantify over 70% of the known mitochondrial proteome over the mass range of 100 to 3,500 kDa. |
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