Most luminal lysosomal proteins are synthesized as precursors containing mannose 
6-phosphate (Man6-P) and a number of recent studies have conducted affinity 
purification of Man6-P containing proteins as a step toward defining the 
composition of the lysosome. Approximately 60 known lysosomal proteins have been 
found in such studies as well as many other Man-6-P glycoproteins, some of which 
represent new lysosomal proteins. The latter are of considerable interest from 
cell-biological and biomedical perspectives, but differentiating between them 
and other proteins remains a significant challenge. The aim of this study was to 
conduct a global analysis of the mammalian Man6-P glycoproteome, implementing 
technical and biostatistical methods to aid in the discovery and validation of 
lysosomal candidates. We purified Man6-P glycoproteins from 17 individual rat 
tissues. To distinguish nonspecific contaminants (i.e., abundant or "sticky" 
proteins that are not fully removed during purification) from specifically 
purified proteins, we conducted a semiquantitative mass spectrometric comparison 
of protein levels in nonspecific mock eluates versus specific affinity 
chromatography eluates to identify those proteins that are specifically 
purified. We identified 60 known lysosomal proteins, representing nearly all 
that are currently known to contain Man-6-P. We also find 136 other proteins 
that are specifically purified but which are not known to have lysosomal 
function. This approach provides a list of candidate lysosomal proteins and also 
provides insights into the relative distribution of Man6-P glycoproteins.