title: Stable isotope labeling with amino acids in cell culture based mass spectrometry approach to detect transient protein interactions using substrate trapping.
abstract: The analysis of protein interactors in protein complexes can yield important insight into protein function and signal transduction. Thus, a reliable approach to distinguish true interactors from nonspecific interacting proteins is of utmost importance for accurate data interpretation. Although stringent purification methods are critical, challenges still remain in the selection of criteria that will permit the objective differentiation of true members of the protein complex from nonspecific background proteins. To address these challenges, we have developed a quantitative proteomic strategy combining stable isotope labeling with amino acids in cell culture (SILAC), affinity substrate trapping, and gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (geLC-MS/MS) protein quantitation. ATP hydrolysis-deficient vacuolar protein sorting-associated protein 4B (Vps4B) was used as the "bait" protein which served as a substrate trap since its lack of ATP hydrolysis enzymatic activity allows the stabilization of its transiently associated interacting proteins. A significant advantage of our approach is the use of our new in-house-developed software program for SILAC-based mass spectrometry quantitation, which further facilitates the differentiation between the bait protein, endogenous bait-interacting proteins, and nonspecific binding proteins based on their protein ratios. The strategy presented herein is applicable to the analysis of other protein complexes whose compositions are dependent upon the ATP hydrolysis activity of the bait protein used in affinity purification studies.
authors: Department of Radiation Oncology, University of Maryland, Baltimore, Maryland, United States. stthomas@som.umaryland.edu
authors: Stefani N
authors: SN
authors: Thomas
authors: Yunhu
authors: Y
authors: Wan
authors: Zhongping
authors: Z
authors: Liao
authors: Phyllis I
authors: PI
authors: Hanson
authors: Austin J
authors: AJ
authors: Yang
references: Nature. 2002 May 23;417(6887):399-403
references: 12000970
references: J Biol Chem. 2010 Nov 12;285(46):35428-38
references: 20805225
references: Dev Cell. 2008 Jul;15(1):62-73
references: 18606141
references: Crit Rev Biochem Mol Biol. 2011 Jun;46(3):216-28
references: 21438726
references: J Mol Biol. 2008 Dec 26;384(4):878-95
references: 18929572
references: J Biol Chem. 2005 Apr 1;280(13):12799-809
references: 15632132
references: Nat Methods. 2006 Dec;3(12):981-3
references: 17072306
references: Nat Rev Mol Cell Biol. 2010 Aug;11(8):556-66
references: 20588296
references: Dev Cell. 2008 Oct;15(4):578-89
references: 18854142
references: Biochemistry. 1999 Nov 9;38(45):14906-15
references: 10555973
references: Hum Mol Genet. 2010 Mar 1;19(5):888-900
references: 20015956
references: Cell Cycle. 2007 Jan 15;6(2):205-17
references: 17314511
references: J Proteomics. 2010 Oct 10;73(11):2198-204
references: 20546956
references: J Cell Biol. 2006 Feb 27;172(5):705-17
references: 16505166
references: FEBS Lett. 2009 Jun 5;583(11):1674-83
references: 19376113
references: J Cell Biol. 2000 Jul 10;150(1):F13-9
references: 10893253
references: J Am Soc Mass Spectrom. 2010 Jan;21(1):127-31
references: 19854660
references: J Cell Biol. 1994 Aug;126(4):945-54
references: 8051214
references: Dev Cell. 2002 Aug;3(2):271-82
references: 12194857
references: Proc Natl Acad Sci U S A. 2004 May 18;101(20):7612-7
references: 15136718
references: EMBO J. 2005 Oct 19;24(20):3658-69
references: 16193069
references: Cell. 1998 Apr 17;93(2):277-87
references: 9568719
references: J Biol Chem. 2009 Oct 9;284(41):27866-27874
references: 19651773
references: Mol Biol Cell. 2008 Jun;19(6):2661-72
references: 18385515
references: J Biol Chem. 2006 Apr 21;281(16):10825-38
references: 16443603
references: J Cell Biol. 2008 Oct 20;183(2):223-39
references: 18936248
references: Mol Cell Proteomics. 2009 Feb;8(2):302-15
references: 18931337
references: Prog Mol Subcell Biol. 1996;16:104-29
references: 8822795
references: Nature. 2009 Mar 12;458(7235):172-7
references: 19234443
references: Anal Chem. 2010 Aug 1;82(15):6643-51
references: 20614870
references: J Biol Chem. 1999 Jul 2;274(27):19316-22
references: 10383442
references: Traffic. 2007 Aug;8(8):1068-79
references: 17547705
references: Semin Cancer Biol. 1993 Feb;4(1):3-12
references: 8448376
references: Cell. 1996 Jun 14;85(6):875-85
references: 8681382
references: Mol Biol Cell. 2004 Feb;15(2):637-48
references: 14617820
references: Mol Cell Proteomics. 2010 Aug;9(8):1650-65
references: 20445003
references: Nature. 2007 Oct 11;449(7163):740-4
references: 17928862
references: Nature. 2007 Oct 11;449(7163):735-9
references: 17928861
references: Curr Genet. 1997 Jun;31(6):469-80
references: 9211789
references: Genomics. 2006 Sep;88(3):333-46
references: 16730941
references: Annu Rev Biophys Biomol Struct. 2006;35:277-98
references: 16689637
references: Mol Cell Proteomics. 2006 Feb;5(2):366-78
references: 16284124
references: EMBO J. 1998 Jun 1;17(11):2982-93
references: 9606181