BACKGROUND: Newborn screening to identify infants with treatable congenital disorders is carried out worldwide. Recent tandem mass spectrometry (MS/MS) applications have markedly expanded the ability to screen for >50 metabolic diseases with a single dried blood spot (DBS). The feature that makes metabolic disorders particularly amenable to screening is the presence of specific small-molecule metabolites. Many treatable disorders such as Wilson disease, however, are characterized by absent or diminished large proteins in plasma or within circulating blood cells, for which there are currently no cost-effective screening methods. METHODS: We developed an assay for quantifying ceruloplasmin (CP) in DBS for newborn screening of Wilson disease. CP-specific peptides from DBS samples digested by trypsin were quantified using isotopically labeled peptide internal standards and liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). RESULTS: The calibration curve was linear from 20 to 95 mg/dL (200-950 mg/L). Intraassay imprecision (mean CV) for CP concentrations of 25, 35, and 55 mg/dL (250, 350, and 550 mg/L) was 9.2%, 10.7%, and 10.2%, respectively. Interassay imprecision for 19 different batches was 8.9%, 5.8%, and 6.9%. A method comparison study on previously tested patient samples for CP gave comparable results with lower limit of quantification, around 0.7 mg/dL (7 mg/L). CONCLUSIONS: Our study supports that newborn screening for Wilson disease is feasible using LC-MS/MS assay for CP quantification in DBS after tryptic digestion. This approach should be applicable to newborn screening for other treatable genetic conditions, such as primary immunodeficiencies, that have large proteins as biomarkers.
BACKGROUND: Newborn screening to identify infants with treatable congenital disorders is carried out worldwide. Recent tandem mass spectrometry (MS/MS) applications have markedly expanded the ability to screen for >50 metabolic diseases with a single dried blood spot (DBS). The feature that makes metabolic disorders particularly amenable to screening is the presence of specific small-molecule metabolites. Many treatable disorders such as Wilson disease, however, are characterized by absent or diminished large proteins in plasma or within circulating blood cells, for which there are currently no cost-effective screening methods. METHODS: We developed an assay for quantifying ceruloplasmin (CP) in DBS for newborn screening of Wilson disease. CP-specific peptides from DBS samples digested by trypsin were quantified using isotopically labeled peptide internal standards and liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). RESULTS: The calibration curve was linear from 20 to 95 mg/dL (200-950 mg/L). Intraassay imprecision (mean CV) for CP concentrations of 25, 35, and 55 mg/dL (250, 350, and 550 mg/L) was 9.2%, 10.7%, and 10.2%, respectively. Interassay imprecision for 19 different batches was 8.9%, 5.8%, and 6.9%. A method comparison study on previously tested patient samples for CP gave comparable results with lower limit of quantification, around 0.7 mg/dL (7 mg/L). CONCLUSIONS: Our study supports that newborn screening for Wilson disease is feasible using LC-MS/MS assay for CP quantification in DBS after tryptic digestion. This approach should be applicable to newborn screening for other treatable genetic conditions, such as primary immunodeficiencies, that have large proteins as biomarkers.
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