Acid hydrolase activities are normally confined within the cell to the lysosome, 
a membrane-delimited cytoplasmic organelle primarily responsible for the 
degradation of macromolecules. However, lysosomal proteins are also present in 
human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a 
modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) 
that normally direct the targeting of these proteins to the lysosome. In this 
study, we purified the Man-6-P glycoforms of proteins from human plasma by 
affinity chromatography on immobilized MPRs and characterized this subproteome 
by two-dimensional gel electrophoresis and by tandem mass spectrometry. As 
expected, we identified many known and potential candidate lysosomal proteins. 
In addition, we also identified a number of abundant classical plasma proteins 
that were retained even after two consecutive rounds of affinity purification. 
Given their abundance in plasma, we initially considered these proteins to be 
likely contaminants, but a mass spectrometric study of Man-6-phosphorylation 
sites using MPR-purified glycopeptides revealed that some proportion of these 
classical plasma proteins contained the Man-6-P modification. We propose that 
these glycoproteins are phosphorylated at low levels by the lysosomal enzyme 
phosphotransferase, but their high abundance results in detection of Man-6-P 
glycoforms in plasma. These results may provide useful insights into the 
molecular processes underlying Man-6-phosphorylation and highlight circumstances 
under which the presence of Man-6-P may not be indicative of lysosomal function. 
In addition, characterization of the plasma Man-6-P glycoproteome should 
facilitate development of mass spectrometry-based tools for the diagnosis of 
lysosomal storage diseases and for investigating the involvement of 
Man-6-P-containing glycoproteins in more widespread human diseases and their 
potential utility as biomarkers.