Purification and proteomic analysis of lysosomal integral membrane proteins.
Lysosomes are essential for normal function of cells. This is best illustrated by the occurrence of greater than 40 lysosomal storage diseases. While the enzymes of the luminal compartment have been widely studied usually in the context of these diseases, the composition of the enveloping membrane has received scant attention. Advances in mass spectrometry and proteomics have laid the necessary groundwork to facilitate investigation of membranes such as those of lysosomes, mitochondria, and other organelles to find novel proteins and novel functions. Pure lysosomes are a prerequisite, and we have successfully identified an abundance of membrane proteins from lysosomes of rat liver. Here, we describe two comparable and easy methods to isolate lysosomes from mouse or rat liver in sufficient quantities for proteomics studies. Also included is a comparison of the soluble, luminal proteins obtained from each of the two preparations separated by 2D immobilized pH gradient (IPG) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
Zhang
Huiwen
H
Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.
Fan
Xiaolian
X
Bagshaw
Rick
R
Mahuran
Don J
DJ
Callahan
John W
JW
D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008247:Lysosomes / Q000737:chemistry* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011205:Povidone; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D020543:Proteome; D012822:Silicon Dioxide; D014357:Trypsin
D007202:Indicators and Reagents; D008565:Membrane Proteins; D010446:Peptide Fragments; D011506:Proteins; D020543:Proteome; C018589:lysosomal proteins; C016039:Percoll; D012822:Silicon Dioxide; D014357:Trypsin; D011205:Povidone