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

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. 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. The genetic bases for numerous human hereditary diseases are well established (1Scriver C.R. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001Google Scholar), but there are some for which the defective genes remain to be identified. For example, in the Online Mendelian Inheritance in Man database (2McKusick V.A. Mendelian Inheritance in Man: a Catalog of Human Genes and Genetic Disorders. 12th Ed. Johns Hopkins University Press, Baltimore1998Google Scholar), there are currently listed over a thousand Mendelian clinical phenotypes of unknown molecular basis. Understanding the molecular basis for disease is essential for genetic screening and developing effective therapy, but identifying individual gene defects can represent a significant challenge. This is particularly true of orphan diseases where patient populations may be small or clinically poorly defined and thus may not be readily amenable to traditional genetics approaches. Proteomics methods provide an alternative route in the investigation of such unsolved genetic diseases and can provide disease gene candidates for further analysis in two different ways. First, comparative proteomics can uncover proteins that are altered in abundance or other properties in specimens from affected individuals, and these may potentially be encoded by the mutant gene. Second, 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 particularly useful in the investigation of lysosomal storage diseases (LSDs) 1The abbreviations used are:LSDlysosomal storage diseaseMPSmucopolysaccharidosisNCLneuronal ceroid lipofuscinosisLINCLlate infantile neuronal ceroid lipofuscinosisANCLadult neuronal ceroid lipofuscinosisMan-6-Pmannose 6-phosphateMPRMan-6-P receptorSGSHN-sulfoglucosamine sulfohydrolase (sulfamidase)CTSDcathepsin DTPP1tripeptidyl-peptidase 1PPT1palmitoyl-protein thioesterase 1LTQlinear trap quadrupoleGPMGlobal Proteome MachineCNScentral nervous systemOMIMOnline Mendelian Inheritance in Man1The abbreviations used are:LSDlysosomal storage diseaseMPSmucopolysaccharidosisNCLneuronal ceroid lipofuscinosisLINCLlate infantile neuronal ceroid lipofuscinosisANCLadult neuronal ceroid lipofuscinosisMan-6-Pmannose 6-phosphateMPRMan-6-P receptorSGSHN-sulfoglucosamine sulfohydrolase (sulfamidase)CTSDcathepsin DTPP1tripeptidyl-peptidase 1PPT1palmitoyl-protein thioesterase 1LTQlinear trap quadrupoleGPMGlobal Proteome MachineCNScentral nervous systemOMIMOnline Mendelian Inheritance in Man (for a review, see Ref. 3Sleat D.E. Jadot proteomics and result from mutations in genes encoding lysosomal and of the primary of the is the of (for a review, see Ref. Press, New Scholar), a of these diseases is an of and other storage the in of affected Most LSD genes have been identified biochemical or genetics but there are some clinically defined LSDs for which the defective genes are not Proteomics approaches have been instrumental in the of gene defects in such human For disease D.E. Jadot of as the gene of and of the gene encoding the in disease Scholar), mutations in genes encoding novel lysosomal proteins that in proteomics In infantile neuronal ceroid the mutant gene was identified as a that was of from brain autopsy specimens of controls but not of affected individuals D.E. of mutations in a lysosomal protein with neuronal ceroid to these clinically defined but unsolved there are individual cases with of lysosomal storage where gene defects have not been identified. are possible for the of a genetic in these cases. First, the and overlap in clinical of with atypical gene defect may be Second, genetic validation may be to mutations of or be from disease may result from mutations in genes that proteins that are not currently to be with this we a comparative analysis of purified mannose from brain autopsy from LSD cases of unsolved or to the molecular basis of disease. Our rationale was that we may identify lysosomal proteins that are altered in of expression in cases compared with controls and that this may provide to the of disease. to this approach is the to lysosomal proteins for Most lysosomal are that a carbohydrate that is by The lysosomal proteins in the and lysosomal proteins the and to an where the of and an can be used to affinity proteins and this numerous of the lysosomal (for a review, see Ref. D.E. Proteomics of the this we that mass proteomics can to a genetic in cases where methods not In to a of for a clinical proteomics approach to this approach was instrumental in the of two lysosomal proteins in which defects may the adult form of a disease of currently this we used spectral protein expression of purified to identify gene defects in LSD of unknown or genetic basis. in a of this approach not identify the gene defect in and this is to be for First, defects in LSDs genes encoding this is not the Second, some mutations may result in but protein and these may be proteins that are the could potentially be in this which was in proteins of relative gene defects may a of a protein be from in or the analysis of of is of more methods of with or spectral counting to such this study for proteomics methods and a trap mass to be identifying or lysosomal defects in eight of of the cases LSD in the of protein the of the that some mutations may for significant expression of of the mutant Thus, in for candidates in cases where defects are not it may be to identify proteins expression from the We this with a analysis of some proteins thioesterase and and even in with confirmed defects in other other proteins in samples, and a significant in such proteins may be and as proteins that in and and these may be further investigation in these cases. is that was in a of disease a of to from this study was the identification of atypical of lysosomal diseases that diagnosed as in is but is of is with and and results in (for a review, see Ref. The Press, The defective gene in the of cases remains to be have been to mutations in neuronal ceroid with thioesterase patients of a neuronal ceroid by in protein thioesterase In this we cases. In the protein approach validated genetics and in it to the identification of the defective an study of an of unknown we purified from a brain from this patient and used to with but defects not apparent D.E. The human brain mannose a mixture of of lysosomal genetics of disease gene candidates in the gene encoding a lysosomal of unknown in the of either an for CLN5 or it was not whether these or of purified that CLN5 was absent in this that these represent the of disease. CLN5 defects a form of with of and into the or of a novel gene encoding a protein in infantile neuronal ceroid Scholar), with of have been of a novel CLN5 in neuronal ceroid CLN5 an atypical neuronal ceroid of For the of disease was of and the patient of the in adult neuronal ceroid of the CLN5 mutations and have been identified Mendelian of these mutations remains to be the that CLN5 was in other cases this and that of CLN5 as an disease provide a that this the is that in this study we CLN5 to be a of the mixture of proteins purified from controls by but we not this protein the by and the by either mass or mass D.E. The human brain mannose a mixture of of lysosomal is possible that CLN5 is the or that the CLN5 peptides are not readily but this result that is well to defects are diagnosed as of the gene and identification of mutations in with that is of and of and into the or of Genetic and clinical in the and that result in have been analysis of identification of novel of gene mutations the in patients with of molecular defects in patients novel adult patient with in the The in the gene is with a clinical phenotype in Scholar), is thus these cases are not diagnosed as is of adult but was not a in this with and of adult have not been with In this mass protein that was in and this to the identification of mutations in the gene. The mutations identified and are analysis of identification of novel of gene mutations the in patients with Scholar), of which is with disease of gene mutations the in patients with The in the gene is with a clinical phenotype in Scholar), which is with is to that are not as a of have been in some patients disease, with some of ceroid Scholar), which may possible with results indicate that clinically defined is not a distinct genetic and the that other cases represent of and In of an of cases as a that either clinical of other or a of neuronal is that the CLN5 and cases to the for as by a with for the CLN5 of the in adult neuronal ceroid and for the This that the genetic of even is more there are in lysosomal that are to a primary defect, and these are we the that the of expression of lysosomal proteins may provide to the of disease. In we a of cases based upon the expression of lysosomal are In cases to be and adult cases to be in genetic basis or and with the of with the is for that the apparent to the cases may lysosomal that result in a and more disease is in the more cases in LSD cases and not to be a in of the cases in this and to be different from the and these of established cases and cases of These may provide useful to the identification of disease genes in cases where the mutant gene is yet and thus may not be as with in D.E. in the gene a lysosomal storage disease with an example, not to be further investigation of this protein may be in the two unsolved cases and with the confirmed the of mass methods for protein expression a approach to the investigation of LSDs of unknown or etiology. In to the identification of gene the of proteomics approaches to different diseases may provide insights into the of these diseases and the of the lysosomal The genetic bases for numerous human hereditary diseases are well established (1Scriver C.R. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001Google Scholar), but there are some for which the defective genes remain to be identified. For example, in the Online Mendelian Inheritance in Man database (2McKusick V.A. Mendelian Inheritance in Man: a Catalog of Human Genes and Genetic Disorders. 12th Ed. Johns Hopkins University Press, Baltimore1998Google Scholar), there are currently listed over a thousand Mendelian clinical phenotypes of unknown molecular basis. Understanding the molecular basis for disease is essential for genetic screening and developing effective therapy, but identifying individual gene defects can represent a significant challenge. This is particularly true of orphan diseases where patient populations may be small or clinically poorly defined and thus may not be readily amenable to traditional genetics approaches. Proteomics methods provide an alternative route in the investigation of such unsolved genetic diseases and can provide disease gene candidates for further analysis in two different ways. First, comparative proteomics can uncover proteins that are altered in abundance or other properties in specimens from affected individuals, and these may potentially be encoded by the mutant gene. Second, 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 particularly useful in the investigation of lysosomal storage diseases (LSDs) 1The abbreviations used are:LSDlysosomal storage diseaseMPSmucopolysaccharidosisNCLneuronal ceroid lipofuscinosisLINCLlate infantile neuronal ceroid lipofuscinosisANCLadult neuronal ceroid lipofuscinosisMan-6-Pmannose 6-phosphateMPRMan-6-P receptorSGSHN-sulfoglucosamine sulfohydrolase (sulfamidase)CTSDcathepsin DTPP1tripeptidyl-peptidase 1PPT1palmitoyl-protein thioesterase 1LTQlinear trap quadrupoleGPMGlobal Proteome MachineCNScentral nervous systemOMIMOnline Mendelian Inheritance in Man1The abbreviations used are:LSDlysosomal storage diseaseMPSmucopolysaccharidosisNCLneuronal ceroid lipofuscinosisLINCLlate infantile neuronal ceroid lipofuscinosisANCLadult neuronal ceroid lipofuscinosisMan-6-Pmannose 6-phosphateMPRMan-6-P receptorSGSHN-sulfoglucosamine sulfohydrolase (sulfamidase)CTSDcathepsin DTPP1tripeptidyl-peptidase 1PPT1palmitoyl-protein thioesterase 1LTQlinear trap quadrupoleGPMGlobal Proteome MachineCNScentral nervous systemOMIMOnline Mendelian Inheritance in Man (for a review, see Ref. 3Sleat D.E. Jadot proteomics and lysosomal storage disease neuronal ceroid infantile neuronal ceroid adult neuronal ceroid mannose sulfohydrolase thioesterase trap Proteome nervous Online Mendelian Inheritance in Man lysosomal storage disease neuronal ceroid infantile neuronal ceroid adult neuronal ceroid mannose sulfohydrolase thioesterase trap Proteome nervous Online Mendelian Inheritance in Man LSDs result from mutations in genes encoding lysosomal and of the primary of the is the of (for a review, see Ref. Press, New Scholar), a of these diseases is an of and other storage the in of affected Most LSD genes have been identified biochemical or genetics but there are some clinically defined LSDs for which the defective genes are not Proteomics approaches have been instrumental in the of gene defects in such human For disease D.E. Jadot of as the gene of and of the gene encoding the in disease Scholar), mutations in genes encoding novel lysosomal proteins that in proteomics In infantile neuronal ceroid the mutant gene was identified as a that was of from brain autopsy specimens of controls but not of affected individuals D.E. of mutations in a lysosomal protein with neuronal ceroid In to these clinically defined but unsolved there are individual cases with of lysosomal storage where gene defects have not been identified. are possible for the of a genetic in these cases. First, the and overlap in clinical of with atypical gene defect may be Second, genetic validation may be to mutations of or be from disease may result from mutations in genes that proteins that are not currently to be with In this we a comparative analysis of purified mannose from brain autopsy from LSD cases of unsolved or to the molecular basis of disease. Our rationale was that we may identify lysosomal proteins that are altered in of expression in cases compared with controls and that this may provide to the of disease. to this approach is the to lysosomal proteins for Most lysosomal are that a carbohydrate that is by The lysosomal proteins in the and lysosomal proteins the and to an where the of and an can be used to affinity proteins and this numerous of the lysosomal (for a review, see Ref. D.E. Proteomics of the In this we that mass proteomics can to a genetic in cases where methods not In to a of for a clinical proteomics approach to this approach was instrumental in the of two lysosomal proteins in which defects may the adult form of a disease of currently basis. this we used spectral protein expression of purified to identify gene defects in LSD of unknown or genetic basis. in a of this approach not identify the gene defect in and this is to be for First, defects in LSDs genes encoding this is not the Second, some mutations may result in but protein and these may be proteins that are the could potentially be in this which was in proteins of relative gene defects may a of a protein be from in or the analysis of of is of more methods of with or spectral counting to such this study for proteomics methods and a trap mass to be identifying or lysosomal defects in eight of of the cases LSD in the of protein the of the that some mutations may for significant expression of of the mutant Thus, in for candidates in cases where defects are not it may be to identify proteins expression from the We this with a analysis of some proteins thioesterase and and even in with confirmed defects in other other proteins in samples, and a significant in such proteins may be and as proteins that in and and these may be further investigation in these cases. is that was in a of disease a of to from this study was the identification of atypical of lysosomal diseases that diagnosed as in is but is of is with and and results in (for a review, see Ref. The Press, The defective gene in the of cases remains to be have been to mutations in neuronal ceroid with thioesterase patients of a neuronal ceroid by in protein thioesterase In this we cases. In the protein approach validated genetics and in it to the identification of the defective an study of an of unknown we purified from a brain from this patient and used to with but defects not apparent D.E. The human brain mannose a mixture of of lysosomal genetics of disease gene candidates in the gene encoding a lysosomal of unknown in the of either an for CLN5 or it was not whether these or of purified that CLN5 was absent in this that these represent the of disease. CLN5 defects a form of with of and into the or of a novel gene encoding a protein in infantile neuronal ceroid Scholar), with of have been of a novel CLN5 in neuronal ceroid CLN5 an atypical neuronal ceroid of For the of disease was of and the patient of the in adult neuronal ceroid of the CLN5 mutations and have been identified Mendelian of these mutations remains to be the that CLN5 was in other cases this and that of CLN5 as an disease provide a that this the is that in this study we CLN5 to be a of the mixture of proteins purified from controls by but we not this protein the by and the by either mass or mass D.E. The human brain mannose a mixture of of lysosomal is possible that CLN5 is the or that the CLN5 peptides are not readily but this result that is well to defects are diagnosed as of the gene and identification of mutations in with that is of and of and into the or of Genetic and clinical in the and that result in have been analysis of identification of novel of gene mutations the in patients with of molecular defects in patients novel adult patient with in the The in the gene is with a clinical phenotype in Scholar), is thus these cases are not diagnosed as is of adult but was not a in this with and of adult have not been with In this mass protein that was in and this to the identification of mutations in the gene. The mutations identified and are analysis of identification of novel of gene mutations the in patients with Scholar), of which is with disease of gene mutations the in patients with The in the gene is with a clinical phenotype in Scholar), which is with is to that are not as a of have been in some patients disease, with some of ceroid Scholar), which may possible with results indicate that clinically defined is not a distinct genetic and the that other cases represent of and In of an of cases as a that either clinical of other or a of neuronal is that the CLN5 and cases to the for as by a with for the CLN5 of the in adult neuronal ceroid and for the This that the genetic of even is more there are in lysosomal that are to a primary defect, and these are we the that the of expression of lysosomal proteins may provide to the of disease. In we a of cases based upon the expression of lysosomal are In cases to be and adult cases to be in genetic basis or and with the of with the is for that the apparent to the cases may lysosomal that result in a and more disease is in the more cases in LSD cases and not to be a in of the cases in this and to be different from the and these of established cases and cases of These may provide useful to the identification of disease genes in cases where the mutant gene is yet and thus may not be as with in D.E. in the gene a lysosomal storage disease with an example, not to be further investigation of this protein may be in the two unsolved cases and with the confirmed the of mass methods for protein expression a approach to the investigation of LSDs of unknown or etiology. In to the identification of gene the of proteomics approaches to different diseases may provide insights into the of these diseases and the of the lysosomal In this we used spectral protein expression of purified to identify gene defects in LSD of unknown or genetic basis. in a of this approach not identify the gene defect in and this is to be for First, defects in LSDs genes encoding this is not the Second, some mutations may result in but protein and these may be proteins that are the could potentially be in this which was in proteins of relative gene defects may a of a protein be from in or the analysis of of is of more methods of with or spectral counting to such this study for proteomics methods and a trap mass to be identifying or lysosomal defects in eight of of the cases LSD in the of protein the of the that some mutations may for significant expression of of the mutant Thus, in for candidates in cases where defects are not it may be to identify proteins expression from the We this with a analysis of some proteins thioesterase and and even in with confirmed defects in other other proteins in samples, and a significant in such proteins may be and as proteins that in and and these may be further investigation in these cases. is that was in a of disease a of disease. to from this study was the identification of atypical of lysosomal diseases that diagnosed as in is but is of is with and and results in (for a review, see Ref. The Press, The defective gene in the of cases remains to be have been to mutations in neuronal ceroid with thioesterase patients of a neuronal ceroid by in protein thioesterase In this we cases. In the protein approach validated genetics and in it to the identification of the defective gene. In an study of an of unknown we purified from a brain from this patient and used to with but defects not apparent D.E. The human brain mannose a mixture of of lysosomal genetics of disease gene candidates in the gene encoding a lysosomal of unknown in the of either an for CLN5 or it was not whether these or of purified that CLN5 was absent in this that these represent the of disease. CLN5 defects a form of with of and into the or of a novel gene encoding a protein in infantile neuronal ceroid Scholar), with of have been of a novel CLN5 in neuronal ceroid CLN5 an atypical neuronal ceroid of For the of disease was of and the patient of the in adult neuronal ceroid of the CLN5 mutations and have been identified Mendelian of these mutations remains to be the that CLN5 was in other cases this and that of CLN5 as an disease provide a that this the is that in this study we CLN5 to be a of the mixture of proteins purified from controls by but we not this protein the by and the by either mass or mass D.E. The human brain mannose a mixture of of lysosomal is possible that CLN5 is the or that the CLN5 peptides are not readily but this result that is well to defects are diagnosed as of the gene and identification of mutations in with that is of and of and into the or of Genetic and clinical in the and that result in have been analysis of identification of novel of gene mutations the in patients with of molecular defects in patients novel adult patient with in the The in the gene is with a clinical phenotype in Scholar), is thus these cases are not diagnosed as is of adult but was not a in this with and of adult have not been with In this mass protein that was in and this to the identification of mutations in the gene. The mutations identified and are analysis of identification of novel of gene mutations the in patients with Scholar), of which is with disease of gene mutations the in patients with The in the gene is with a clinical phenotype in Scholar), which is with is to that are not as a of have been in some patients disease, with some of ceroid Scholar), which may possible with These results indicate that clinically defined is not a distinct genetic and the that other cases represent of and In of an of cases as a that either clinical of other or a of neuronal is that the CLN5 and cases to the for as by a with for the CLN5 of the in adult neuronal ceroid and for the This that the genetic of even is more In there are in lysosomal that are to a primary defect, and these are we the that the of expression of lysosomal proteins may provide to the of disease. In we a of cases based upon the expression of lysosomal are In cases to be and adult cases to be in genetic basis or and with the of with the is for that the apparent to the cases may lysosomal that result in a and more disease is in the more cases in LSD cases and not to be a in of the cases in this and to be different from the and these of established cases and cases of These may provide useful to the identification of disease genes in cases where the mutant gene is yet and thus may not be as with in D.E. in the gene a lysosomal storage disease with an example, not to be further investigation of this protein may be in the two unsolved cases and with the confirmed In the of mass methods for protein expression a approach to the investigation of LSDs of unknown or etiology. In to the identification of gene the of proteomics approaches to different diseases may provide insights into the of these diseases and the of the lysosomal with autopsy from and was essential for this We and as well as of the and for in this We the Human and the and the and for for specimens and clinical with with

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