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Submitted on October 18, 2005
CABM, Piscataway, NJ 08854
Corresponding Author: sleat{at}cabm.rutgers.edu
Most newly synthesized soluble lysosomal proteins contain mannose 6-phosphate (Man6-P), a specific carbohydrate modification that is recognized by Man6-P receptors (MPRs) that direct targeting to the lysosome. A number of proteomic studies have focused on lysosomal proteins, exploiting the fact that Man6-P-containing forms can be purified by affinity chromatography on immobilized MPRs. These studies have identified many known lysosomal proteins as well as many proteins not previously classified as lysosomal. The latter are of considerable biological interest with potential implications for lysosomal function and as candidates for lysosomal storage diseases of unknown etiology. However, a significant problem in interpreting the biological relevance of such proteins has been in distinguishing true Man6-P glycoproteins from simple contaminants and from proteins associated with true Man6-P glycoproteins (e.g. protease inhibitors and lectins). In this report, we describe a mass-spectrometric approach to the verification of Man6-phosphorylation based upon LC-MS of MPR-purified proteolytic glycopeptides. This has provided a useful tool in validating novel MPR-purified proteins as true Man6-P glycoproteins and has also allowed identification of low-abundance components not observed in the analysis of the total Man6-P glycoprotein mixture. In addition, this approach has allowed the global mapping of 99 Man6-phosphorylation sites from 44 known lysosomal proteins purified from mouse and human brain. This information is likely to provide useful insights into protein determinants for this modification and may be of significant value in protein engineering approaches designed to optimize protein delivery to the lysosome in therapeutic applications such as gene and enzyme-replacement therapies.
Revised on December 13, 2005
Accepted on January 5, 2006
Identification of sites of mannose 6-phosphorylation on lysosomal proteins
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