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Maria Cecilia Della Valle
Biochemist - focus in Biotechnology
University of Buenos Aires
Thesis Advisor: Peter Lobel, PhD
Graduate Program: Cellular and Molecular Pharmacology
Wednesday, June 17, 2009
Lysosomes play an essential role in the degradation of macromolecules. There are over forty known lysosomal storage disorders (LSDs) that are caused by mutations in genes encoding lysosomal proteins. This group of diseases is frequently characterized by neurodegeneration and shortened lifespan. For some LSDs, the genetic basis remains unknown. Finding of new lysosomal proteins provides new candidates for these diseases and also contributes to a deeper understanding of the biology of the lysosome. Several proteomic studies of lysosome-enriched preparations have provided lysosomal candidates but distinguishing true lysosomal proteins from contaminants remains challenging.
In the present work we carried out the study of the subcellular localization of EPDR, a candidate but non-validated lysosomal protein. We used a biochemical approach that involved the generation of an anti-EPDR antibody and development of a novel subcellular fractionation approach for the localization of lysosomal enzymes in brain. This work allowed us to demonstrate that EPDR truly is a resident lysosomal protein.
A rate-limiting step in these studies was the generation of high quality antibody reagents, which required over a year of dedicated effort (generation and purification of recombinant protein, immunization of rabbits and characterization of antibodies). We thus evaluated the use of quantitative mass spectrometry (MS) combined with subcellular fractionation as a general approach to identify and validate components of the lysosomal proteome. Rat liver lysosome-enriched preparations from controls and animals treated to alter the buoyant density of lysosomes were further fractionated by isopycnic sucrose density gradient centrifugation. Peptides derived from gradient fractions were chemically labeled with stable isotopes (iTRAQ) followed by two-dimensional chromatography and MALDI-TOF/TOF mass spectrometric analysis. This approach allowed us to measure the relative abundance of proteins across the density gradients to generate protein distribution profiles. The quantitative MS results for known lysosomal enzymes were in agreement with biochemical (enzymatic) assays.
These results confirmed that quantitative mass spectrometry can be combined with subcellular fractionation to confidently measure the distribution of lysosomal proteins in complex subcellular fractions, and has allowed for the identification of several new candidates for lysosomal proteins.