Most newly synthesized soluble lysosomal proteins contain mannose 6-phosphate 
(Man-6-P), a specific carbohydrate modification that is recognized by Man-6-P 
receptors (MPRs) that direct targeting to the lysosome. A number of proteomic 
studies have focused on lysosomal proteins, exploiting the fact that 
Man-6-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 Man-6-P glycoproteins from simple 
contaminants and from proteins associated with true Man-6-P glycoproteins (e.g. 
protease inhibitors and lectins). In this report, we describe a mass 
spectrometric approach to the verification of Man-6-phosphorylation based upon 
LC-MS of MPR-purified proteolytic glycopeptides. This provided a useful tool in 
validating novel MPR-purified proteins as true Man-6-P glycoproteins and also 
allowed identification of low abundance components not observed in the analysis 
of the total Man-6-P glycoprotein mixture. In addition, this approach allowed 
the global mapping of 99 Man-6-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.