Abstract
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.
Highlights
Lysosomes are membrane-delimited organelles that are responsible for the degradation of macromolecules delivered via various cellular pathways including endocytosis, phagocytosis, and autophagy
The first step was to prepare light mitochondrial (L) differential centrifugation fractions from individual control and Triton WR-1339 treated rats and further fractionate these using sucrose density gradient centrifugation
In addition to representing samples derived from different animals, Experiment I and II differed in terms of which Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) 8-plex reagent was used to label fractions from the different gradient fractions and on the collision energies used for MS/MS
Summary
Lysosomes are membrane-delimited organelles that are responsible for the degradation of macromolecules delivered via various cellular pathways including endocytosis, phagocytosis, and autophagy. Alterations in lysosomal function have been linked with more widespread human disorders, including cancer [2] and neurodegenerative disorders such as Alzheimer and Parkinson diseases [3,4,5], there is a growing interest in understanding the function of this organelle One approach to this lies in characterizing its proteome. A differential centrifugation fraction enriched in lysosomes is subjected to further fractionation methods, typically isopycnic density gradient centrifugation, and the distribution of the protein of interest again is compared with that of known markers. This density shift is a specific hallmark of lysosomal proteins that has been used in the case-by-case verification of lysosomal candidates identified in proteomics experiments [12,13,14,15,16,17]
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