The lysosomal matrix is estimated to contain about 50 different proteins. Most 
of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate 
receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive 
proteome analysis of MPR-binding proteins from mouse. Mouse embryonic 
fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to 
secrete the lysosomal matrix proteins. Secretions of these cells were affinity 
purified using an affinity matrix derivatized with MPR46 and MPR300. In the 
protein fraction bound to the affinity matrix and eluted with mannose 
6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the 
lysosomal matrix and 4 non-lysosomal contaminants were identified by mass 
spectrometry after separation by two-dimensional gel electrophoresis or by 
multidimensional protein identification technology. For 3 of the candidate 
proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible 
serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could 
verify that C-terminally tagged forms bound in an M6P-dependent manner to an 
MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence 
these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix 
proteins.