title: Detection of mutant protein in complex biological samples: glucocerebrosidase mutations in Gaucher's disease.
abstract: We report a sensitive method to detect point mutations in proteins from complex samples. The method is based on surface-enhanced laser desorption/ionization time-of-flight (SELDI-ToF) MS but can be extended to other MS platforms. The target protein in this study is the lysosomal enzyme glucocerebrosidase (GC), the key enzyme in Gaucher's disease. Deficiency of GC activity results in accumulation of glucosylceramide in macrophages. The relationship between GC genotypes and Gaucher's patient phenotypes is not strict. The possibility to measure protein levels of GC in clinical samples may provide deeper insight into the phenomenology of Gaucher's disease. For this purpose, GC was isolated in a single enrichment step through interaction with an immobilized monoclonal antibody, 8E4. After on-chip digestion of the antibody-antigen complex with trypsin, a total of 25 GC peptides were identified (sequence coverage approximately 60%), including several peptides containing mutated amino acid residues. The described methodology allows mutational analysis on the protein level, directly measured on complex biological samples without the necessity of elaborate purification procedures.
authors.affiliation: Clinical Proteomics Group, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
authors.forename: Boris
authors.initials: B
authors.lastname: Bleijlevens
authors.forename: Mariëlle J
authors.initials: MJ
authors.lastname: van Breemen
authors.forename: Wilma E
authors.initials: WE
authors.lastname: Donker-Koopman
authors.forename: Chris G
authors.initials: CG
authors.lastname: de Koster
authors.forename: Johannes M F G
authors.initials: JM
authors.lastname: Aerts
mesh_terms: D000595:Amino Acid Sequence; D002849:Chromatography, Gas; D005776:Gaucher Disease / Q000201:enzymology*; D005962:Glucosylceramidase / Q000737:chemistry / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D005962:Glucosylceramidase