Classification of Subcellular Location by Comparative Proteomic Analysis of Native and Density-shifted Lysosomes

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

Proteomics Insights into Autophagy

“…Using iTRAQ labeling and two dimensional peptide separation involving strong cation exchange and reverse phase chromatography, Della Valle et al. also report the MALDI‐TOF proteomics identification of high confident lysosomal proteins using an isolation strategy involving the combination of differential centrifugation with sucrose density centrifugation following treatment with Triton‐WR1339 . Among their high confident proteins were cathepsin D (CTSD), classical lysosomal acid phosphatases (ACP2, ACP5), and lysosomal associated membrane protein 2 (LAMP2).…”

Bioanalysis of Eukaryotic Organelles

“…274 Unfortunately, the discovery and identification of lysosomal proteins has been difficult due to the presence of other contaminating organelles (such as mitochondria) in the lysosomal fraction when using conventional s ubcellular fractionation techniques. Using lysosomal density shifting techniques (treatment with either Triton WR-1339 275 or progesterone 276 ), investigators have been able to assign proteins to the lysosome in Wistar rat livers and human hepatocellular carcinoma (HepG2) cells, respectively, that were n ot previously assigned to that organelle. In addition, they were able to identify proteins associated with other organelles in the same fraction that were previously assigned to the lysosome using a quadratic discriminant analysis.…”

“…In addition, they were able to identify proteins associated with other organelles in the same fraction that were previously assigned to the lysosome using a quadratic discriminant analysis. 275 More specifically, if levels of a given protein change in parallel with the organelle marker(s) (e.g. Lamp2 is a marker for lysosomes), it strongly suggests that the protein is localized with that particular o rganelle.…”

Subcellular Trafficking of Mammalian Lysosomal Proteins: An Extended View

“…The subsequent findings that these enzymes co-distribute in rat liver subcellular fractions, and that their distribution profile (i.e., total amount, and enrichment level over total proteins in each fraction) differs from those reported for proteins located in other cellular structures led to the discovery of lysosomes ([ 1 ], reviewed by Sabatini and Adesnik [ 2 ]). Today, proteomic analyses have revealed that the lumen of lysosomes contains approximately 60 different acid hydrolases, and that the lysosomal membrane is spanned by many transmembrane proteins [ 3 , 4 , 5 , 6 , 7 ]. These include structural proteins, a transmembrane vATPase complex that generates an intraluminal acidic environment in which acid hydrolases are active, as well as a large set of transporters that transfer the enzyme degradation products in the cytosol.…”