This chapter describes the process of production, purification, separation, and 
mass spectrometry identification of soluble lysosomal proteins. The rationale 
for purification of these proteins resides in their characteristic sugar, the 
mannose-6-phosphate (M6P), which allows an easy purification by affinity 
chromatography on immobilized M6P receptor (MPR). The secretion of M6P proteins 
(essentially soluble lysosomal proteins) from cells in culture is induced by 
adding a weak base in the culture medium. Secreted proteins are ammonium sulfate 
precipitated, dialyzed, and loaded onto the immobilized MPR column. After 
specific elution and collection of the M6P proteins, these are resolved by 
either bidimensional or monodimensional gel electrophoresis (designated as 2-DE 
or 1-DE, respectively). Mass spectrometry analysis is performed on spots excised 
from the 2-DE gel, or on discrete bands covering altogether the whole length of 
the 1-DE gel lane: these spots or bands are in-gel digested with trypsin and 
protein identification is obtained, thanks to peptide mass fingerprints 
[provided by analysis of the digests by matrix-assisted laser desorption 
ionization-mass spectrometry (MALDI-MS)] or peptide amino acid sequences 
(provided by analysis of the digests by the coupling between liquid 
chromatography and tandem mass spectrometry, LC-MS/MS).