The mannose 6-phosphate glycoprotein proteome

Abstract

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.

Figure 1

Distribution of known lysosomal proteins based upon expression profiling based upon number of EST counts. Panel A. Expression profiles of 63 known soluble lysosomal proteins were determined by the detection of transcripts in 45 different tissue samples in the human Unigene Build #247 database (http://www.ncbi.nlm.nih.gov/UniGene/). Transcript count data are shown in Supplemental Table 1. Panel B. Sum of transcripts per million for each lysosomal protein in all tissues as a function of the number of tissues in which ESTs corresponding to each protein were found.

Figure 2

Relationship between amount of sample analyzed and number of spectral counts observed. Panel A. Increasing amounts of a tryptic digest of a rat liver preparation were analyzed in duplicate by LC-MS and the total number of spectral counts determined for each sample. Error bars represent range. Panel B. For each tissue sample, the ratio of spectral counts in specific versus mock eluates is plotted against the ratio of protein analyzed in the two samples.

Figure 3

Enrichment factors in specific versus mock eluates obtained for proteins identified by affinity chromatography on immobilized MPRs. The log₂ of the ratio of spectral counts in the specific and mock eluates (SC_M6P and SC_MOCK, respectively) is plotted with bars representing the upper and lower 95% confidence indices for known lysosomal proteins (Panel A) or proteins not currently classified as lysosomal (Panel B). The lower confidence index all but one of the known lysosomal proteins is greater than 2.75 (log₂=1.5) and this threshold is plotted as a dotted line. Plots represent: green error bars, proteins that achieve this threshold; blue error bars, proteins that do not achieve this threshold but which are significantly enriched in the specific eluate (lower 95% confidence interval is greater than 1 (log₂=0)); grey error bars, proteins that cannot be classified (95% confidence interval includes SC_M6P/SC_MOCK =1); and red error bars, proteins that are significantly depleted in the specific eluate (upper 95% confidence interval is less than 1 (log₂=0)). For graphical representation, fold-enrichments that are greater than 16 or less that 1/16 are arbitrarily assigned to be 16 (a log₂ value of 4) or 1/16 (a log₂ value of −4) respectively. Proteins that not are of rodent origin are not shown.

Figure 4

Distribution of affinity purified proteins. The number of tissues is shown in which lysosomal proteins reported to contain Man6-P (Panel A) and the remaining proteins (Panel B) were identified in Man6-P eluates.

Figure 5

Tissue distribution as a function of enrichment in the specific eluate. The number of tissues is shown for which proteins categorized as unclassified (Panel A), non-lysosomal (Panel B), secondary lysosomal candidates (Panel C) or primary lysosomal candidates (Panel D) were identified in Man6-P eluates.

Figure 6

Widely-distributed proteins that are significantly enriched in the specific eluate. These proteins were identified in Man6-P eluates from >12 different tissues. The log₂ of the ratio of spectral counts in the specific and mock eluates (SC_M6P and SC_MOCK, respectively) is plotted with bars representing the upper and lower 95% confidence indices. Plots in green represent primary lysosomal candidates (i.e., where the lower 95% confidence index is greater than 1.5, a threshold that is plotted as a dotted line) and plots in blue indicate secondary lysosomal candidates (i.e., proteins that are significantly enriched in the specific eluate).