Classification of subcellular location by comparative proteomic analysis of native and density-shifted lysosomes.
One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies.
Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Maria Cecilia
MC
Della Valle
David E
DE
Sleat
Haiyan
H
Zheng
Dirk F
DF
Moore
Michel
M
Jadot
Peter
P
Lobel
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D016002:Discriminant Analysis; D057075:Enzyme Assays; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D015388:Organelles / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013046:Specific Gravity; D013347:Subcellular Fractions / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D053719:Tandem Mass Spectrometry
D018528:ATP-Binding Cassette Transporters; C491540:Abcb6 protein, rat; D015415:Biomarkers; D020543:Proteome; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1
Am J Physiol Cell Physiol. 2008 Feb;294(2):C579-90
18160489
Biochem Biophys Res Commun. 2008 May 2;369(2):369-75
18279659
J Proteomics. 2009 Feb 15;72(1):23-33
19068244
Mol Cell Proteomics. 2007 May;6(5):767-80
17272266
Bioinformatics. 2004 Jun 12;20(9):1466-7
14976030
Nucleic Acids Res. 2010 Jan;38(Database issue):D800-5
19892824
J Proteome Res. 2009 Nov;8(11):5347-55
19754192
J Biol Chem. 2001 Oct 19;276(42):38388-93
11507097
Nature. 2006 Oct 5;443(7111):586-9
17006453
EMBO J. 1992 Mar;11(3):813-9
1547784
Mol Cell Proteomics. 2005 Jul;4(7):945-57
15851778
Cell. 2008 Jul 11;134(1):112-23
18614015
Proteomics. 2007 Oct;7(20):3651-60
17880003
Mol Cell Proteomics. 2005 Feb;4(2):133-43
15579476
Biochim Biophys Acta. 2003 Sep 23;1651(1-2):116-23
14499595
J Proteome Res. 2008 Jul;7(7):3010-21
18507433
Biochim Biophys Acta. 2006 Dec;1760(12):1903-13
16996694
Neuron. 2008 Oct 23;60(2):247-57
18957217
Traffic. 2007 Dec;8(12):1676-1686
17897319
Mol Cell Proteomics. 2006 Apr;5(4):686-701
16399764
Methods Enzymol. 1990;182:203-25
2156127
J Cell Biol. 1993 Feb;120(4):897-908
8432730
J Biol Chem. 1982 Aug 10;257(15):8945-52
7096343
N Engl J Med. 2009 Oct 22;361(17):1651-61
19846850
Biochemistry. 2007 Aug 21;46(33):9443-52
17661442
Acta Microbiol Immunol Hung. 2005;52(3-4):309-22
16400872
Hum Mol Genet. 2008 Nov 1;17(21):3303-17
18703498
J Biol Chem. 1996 Aug 9;271(32):19191-8
8702598
Biochem J. 1964 Jul;92(1):184-205
4378797
Proteomics Clin Appl. 2007 Sep;1(9):1134-46
21136763
J Biol Chem. 2000 Jun 9;275(23):17536-40
10837493
FEBS Lett. 2006 Oct 16;580(24):5747-52
17007843
J Proteome Res. 2006 Nov;5(11):3135-44
17081065
Mol Cell Proteomics. 2006 Oct;5(10):1942-56
16709564
Biochem J. 2009 Jul 29;422(1):83-90
19489740
Calcif Tissue Int. 2009 Dec;85(6):465-83
19915788
Mass Spectrom Rev. 2004 Nov-Dec;23(6):393-442
15290709
Biochem J. 2007 Mar 15;402(3):449-58
17105447
J Biol Chem. 2006 Nov 17;281(46):35436-45
16954209
J Cell Biol. 1968 May;37(2):482-513
4297786
Traffic. 2009 Nov;10(11):1711-21
19686298
Biochim Biophys Acta. 2009 Apr;1793(4):625-35
18977398
J Neurochem. 1999 Aug;73(2):700-11
10428067
Biochemistry. 2010 Jun 15;49(23):4715-23
20450191
Biochem J. 1955 Aug;60(4):604-17
13249955
J Cell Biol. 1991 Jan;112(1):81-94
1846001
Biochem J. 2006 Apr 1;395(1):39-47
16367739
Placenta. 1998 Nov;19(8):643-54
9859869
Proc Natl Acad Sci U S A. 2009 May 5;106(18):7619-23
19372378
Proc Natl Acad Sci U S A. 1995 May 9;92(10):4299-303
7753801
Science. 2000 Dec 22;290(5500):2298-301
11125141
Proc Natl Acad Sci U S A. 2008 Oct 28;105(43):16590-5
18940929
Proteomics. 2010 Nov;10(22):4053-76
20957757
Mol Cell Proteomics. 2008 Jan;7(1):58-70
17848585
J Cell Biol. 1971 Jul;50(1):20d-55d
4327465
J Cell Sci. 2006 Apr 15;119(Pt 8):1494-503
16537643
Biochem Biophys Res Commun. 2006 Jun 16;344(4):1186-91
16650381
J Cell Sci. 2004 Sep 15;117(Pt 20):4705-15
15331635
Anal Biochem. 2008 Mar 1;374(1):31-40
18082124
Fertil Steril. 2006 Aug;86(2):433-9
16769060
Cell. 2010 Jun 25;141(7):1146-58
20541250
Methods Enzymol. 1984;105:121-6
6727660