The lysosome is a membrane delimited cytoplasmic organelle that contains at 
least 50 hydrolytic enzymes and associated cofactors. The biomedical importance 
of these enzymes is highlighted by the many lysosomal storage disorders that are 
associated with mutations in genes encoding lysosomal proteins, and there is 
also evidence that lysosomal activities may be involved in more widespread human 
diseases. The aim of this study was to characterize the human brain lysosomal 
proteome with the goal of establishing a reference map to investigate human 
diseases of unknown etiology and to gain insights into the cellular function of 
the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate 
modification used for targeting resident soluble lysosomal proteins to the 
lysosome, were affinity-purified using immobilized Man6-P receptor. 
Fractionation by two-dimensional electrophoresis resolved a complex mixture 
comprising approximately 800 spots. Constituent proteins in each spot were 
identified using a combination of matrix-assisted laser 
desorption/ionization-time of flight mass spectrometry (both peptide mass 
fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic 
digests and N-terminal sequencing. In a complementary analysis, we also analyzed 
a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. 
In total, 61 different proteins were identified. Seven were likely contaminants 
associated with true Man6-P glycoproteins. Forty-one were known lysosomal 
proteins of which 11 have not previously been reported to contain Man6-P. An 
additional nine proteins were either uncharacterized or proteins not previously 
reported to have lysosomal function. We found that the human brain 
Man6-P-containing lysosomal proteome is highly complex and contains more 
proteins with a much greater number of individual isoforms than found in 
previous studies of Man6-P glycoproteomes.