Haiqing Yu1, Joann J. Lu1, Wei Rao1, and Shaorong Liu1
- Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA
Density gradient centrifugation is widely utilized for various high purity sample preparations, and density gradient ultracentrifugation (DGU) is often used for more resolution-demanding purification of organelles and protein complexes. Accurately locating different isopycnic layers and precisely extracting solutions from these layers play a critical role in achieving high-resolution DGU separations. In this technique note, we develop a DGU procedure by freezing the solution rapidly (but gently) after centrifugation to fix the resolved layers and by slicing the frozen solution to fractionate the sample. Because the thickness of each slice can be controlled to be as thin as 10 micrometers, we retain virtually all the resolution produced by DGU. To demonstrate the effectiveness of this method, we fractionate complex V from HeLa mitochondria using a conventional technique and this freezing-slicing (F-S) method. The comparison indicates that our F-S method can reduce complex V layer thicknesses by ~40%. After fractionation, we analyze complex V proteins directly on a matrix assisted laser desorption/ionization, time-of-flight mass spectrometer. Twelve out of fifteen subunits of complex V are positively identified. Our method provides a practical protocol to identify proteins from complexes, which is useful to investigate biomolecular complexes and pathways in various conditions and cell types.
CovalX Technology Used (Click each option to learn more)
Ice slices from the F-S method of mitochondrial extraction were thawed on ice and then filtered to remove sucrose and salts using an ultra filter column. Next, the sample was suspended in DDI water that contained 0.05% w/v lauryl maltoside and then mixed with matrix. The mixture was placed on a target and analyzed using a mass spectrometer that had been modified with a CovalX HM2 detection system. The data was then analyzed using the CovalX Complex Tracker software. Through the use of MALDI-TOF-MS, researchers were able to positively identify 12 of 15 subunits found in mitochondrial complex V.