Capitalizing Resolving Power of Density Gradient Ultracentrifugation by Freezing and Precisely Slicing Centrifuged Solution: Enabling Identification of Complex Proteins from Mitochondria by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.
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.
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.
Haiqing
H
Yu
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.
Joann J
JJ
Lu
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.
Wei
W
Rao
Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.
Shaorong
0000-0002-4602-0483
S
Liu
J Chromatogr A. 2015 Sep 25;1413:33-46
26306914
Appl Microbiol. 1966 Jul;14(4):687-9
5927055
Anal Biochem. 1974 Jan;57(1):89-92
4817513
EMBO J. 2000 Sep 15;19(18):4895-902
10990453
J Vis Exp. 2011 Feb 24;(48):null
21403626
Nature. 1961 Jan 21;189:220-1
13773838
Anal Biochem. 1979 Aug;97(1):72-6
484844
Science. 1968 Jul 26;161(3839):386
5661297
J Vis Exp. 2016 Jan 05;(107):
26780239
J Biol Chem. 2016 Feb 19;291(8):4178-84
26698328
J Biol Chem. 1995 Mar 3;270(9):4288-92
7876188
Anal Biochem. 1982 Feb;120(1):208-10
7091655
Mol Cell. 2008 Nov 21;32(4):529-39
19026783
Eur J Pharm Biopharm. 2016 Jan;98 :1-8
26545617
J Virol. 2015 Oct;89(20):10359-70
26246569
Anal Biochem. 1973 May;53(1):339-41
4711111
Nature. 2007 Dec 13;450(7172):991-1000
18075578
Histochem Cell Biol. 2015 Oct;144(4):331-46
26105026
Methods Enzymol. 1995;260:190-202
8592444
Anal Biochem. 2009 Jun 15;389(2):143-9
19348780
Anal Chem. 2014 Nov 18;86(22):11018-23
25325436
Anal Biochem. 1966 Jun;15(3):539-41
5334068
Nat Nanotechnol. 2010 Jun;5(6):443-50
20453856
Anal Biochem. 1971 Apr;40(2):490-3
5551561
J Am Chem Soc. 2007 Jul 4;129(26):8058-9
17552526
J Biol Chem. 2002 Aug 30;277(35):32157-64
12065580