Mass Spectrometry-based Protein Profiling to Determine the Cause of Lysosomal Storage Diseases of Unknown Etiology

David E Sleat,Lin Ding,Shudan Wang,Caifeng Zhao,Yanhong Wang,Winnie Xin,Haiyan Zheng,Dirk F Moore,Katherine B Sims

doi:10.1074/mcp.m900122-mcp200

Abstract

Diagnosis of lysosomal storage diseases (LSDs) can be problematic in atypical cases where clinical phenotype may overlap with other genetically distinct disorders. In addition, LSDs may result from mutations in genes not yet implicated in disease. Thus, there are individuals that are diagnosed with apparent LSD based upon clinical criteria where the gene defect remains elusive. The objective of this study was to determine whether comparative proteomics approaches could provide useful insights into such cases. Most LSDs arise from mutations in genes encoding lysosomal proteins that contain mannose 6-phosphate, a carbohydrate modification that acts as a signal for intracellular targeting to the lysosome. We purified mannose 6-phosphorylated proteins by affinity chromatography and estimated relative abundance of individual proteins in the mixture by spectral counting of peptides detected by tandem mass spectrometry. Our rationale was that proteins that are decreased or absent in patients compared with controls could represent candidates for the primary defect, directing biochemical or genetics studies. On a survey of brain autopsy specimens from 23 patients with either confirmed or possible lysosomal disease, this approach identified or validated the genetic basis for disease in eight cases. These results indicate that this protein expression approach is useful for identifying defects in cases of undiagnosed lysosomal disease, and we demonstrated that it can be used with more accessible patient samples, e.g. cultured cells. Furthermore this approach was instrumental in the identification or validation of mutations in two lysosomal proteins, CLN5 and sulfamidase, in the adult form of neuronal ceroid lipofuscinosis.

Highlights

Diagnosis of lysosomal storage diseases (LSDs) can be problematic in atypical cases where clinical phenotype may overlap with other genetically distinct disorders
In classical late infantile neuronal ceroid lipofuscinosis (LINCL), the mutant gene product was identified as a spot that was present on twodimensional gels of extracts from brain autopsy specimens of controls but not of affected individuals [7]
Strategy for the Identification of Gene Defects—The rationale underlying this study was that a mass spectrometric proteomics analysis of lysosomal proteins could reveal those that are absent, diminished, or otherwise altered in LSD cases compared with controls

Summary

Introduction

Diagnosis of lysosomal storage diseases (LSDs) can be problematic in atypical cases where clinical phenotype may overlap with other genetically distinct disorders. Descriptive proteomics can identify novel proteins with known or predicted properties or expression patterns that may associate them with diseases of unknown etiology Such approaches can be applicable to small cohorts or even individual cases and have been useful in the investigation of lysosomal storage diseases (LSDs) (for a review, see Ref. 3). In classical late infantile neuronal ceroid lipofuscinosis (LINCL), the mutant gene product was identified as a spot that was present on twodimensional gels of extracts from brain autopsy specimens of controls but not of affected individuals [7] In addition to these clinically defined but unsolved diseases, there are individual cases with histopathological evidence of lysosomal storage where gene defects have not been identified.

Objectives

Results

Conclusion