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Assay for Biotinidase Deficiency by Tandem Mass Spectrometry

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Assay for Biotinidase Deficiency by Tandem Mass Spectrometry Assay for Biotinidase Deficiency by Tandem Mass Spectrometry Blas Cerda and Harry Sterling 2004 Newborn and Genetic Testing Symposium May 3-6, Atlanta, Georgia 1 Biotinidase Deficiency Ø Biotinidase • Recycles biotin from biocytin and small biotinylated peptides • Biocytin forms upon proteolytic degradation of biotin-dependant carboxylases Ø Biotinidase Deficiency • Inherited metabolic disorder • ~ 1 in 60,000 incidence rate »Profound deficiency: Less than 10% the normal activity »Partial deficiency: 10-30% the normal activity 2 Biotin Cycle Lysine BIOTINIDASE Biotin Biocytin Carboxylases Inactive proteolysis carboxylases Active carboxylases 3 Biotin Cycle O Biocytin HN NH O NH2 O N S H OH H2O BIOTINIDASE O HN NH O NH2 ++ O OH H2N S OH Biotin Lysine 4 Biotinidase Deficiency Ø Current Methods • Colorimetric • N-Biotinyl-p-aminobenzoate (B-PAB) O HN NH O O N S H OH B-PAB BIOTIN ++ PAB BIOTINIDASE Color development 5 Biotinidase Deficiency Ø Current Methods Ø Fluorometric • N-Biotinyl-6-aminoquinoline (B-6AQ) O N HN NH O N S H B-6AQ BIOTIN ++ 6AQ BIOTINIDASE Fluorescence measurement 6 Biotinidase Deficiency Ø Current Methods • Fluorometric • Biocytin O HN NH O NH2 O N S H OH Biocytin BIOTIN ++ lysine Derivatization BIOTINIDASE Fluorescence measurement 7 Biotinidase Deficiency by MS/MS MS/MS Enzyme MS/MS Enzyme Lysine activity assay BIOTINIDASE Biocytin Biotin Carboxylases Inactive proteolysis carboxylases Add exogenous substrate: Follow substrate to Active carboxylases product conversion by Active carboxylases MS/MS 8 Biotinidase Activity MS/MS Assay We are researching the possibility of developing a MS/MS assay for measuring Biotinidase activity in blood spots O Biocytin HN NH O NH2 Add O N S H OH MS/MS O BIOTINIDASE HN NH NH2 O O ++ H2N OH OH S Lysine Biotin 9 Biotinidase Activity MS/MS Assay Add Buffer Add We are investigating the substrate possibility of labeled biocytin and product IS Stop DBS reaction Punch MS/MS of biocytin and lysine Incubate Currently; lysine : biocytin ratio Low activity = smaller ratio MS/MS High activity = larger ratio 10 Preliminary Linearity 2.5 Dilution LOW HIGH X axis 100 0 0 2 80 20 20 60 40 40 40 60 60 1.5 20 80 80 0 100 100 1 y = 0.0197x + 0.2225 R2 = 0.9968 Lysine/Biocytin 0.5 0 0 20 40 60 80 100 Dilution Factor (%) 11 Lysine/Biocytin Precision 12 replicates per 15% experimental condition 12% CV 9% 6% 3% 0% 1 3 5 7 9 11 13 15 17 19 21 23 25 27 ave Assay Condition 12 Biotinidase Activity 1.20 1.00 0.80 0.60 0.40 Lysine/Biocytin Ratio 0.20 0.00 A B C D E F Endo Calibrator 13 Biotinidase Activity + AAAC MS/MS Assay Add Buffer Add substrate Optimized assay for both Incubate Stop DBS reaction Punch Measure: Biocytin-Lysine conversion + MS/MS AAAC All in the same well 14 Combined assay vs. fluorescent assay BIOTINIDASE ACTIVITY 300 225 150 Normal 75 Deficient e.u. nmol/(dL min) 0 A B C D E F CALIBRATORS MS/MS NCS 15 Biotinidase Activity + AAAC MS/MS Assay AA Correlation to AAAC only assay AA SLOPE R2 ALA 1.04 0.9916 ARG 1.11 0.9975 CIT 1.13 0.9912 GLY 1.36 0.9836 LEU 1.14 0.9987 MET 1.00 0.9989 ORN 1.02 0.9951 PHE 0.99 0.9949 TYR 1.10 0.9917 VAL 1.04 0.9836 MEAN 1.09 0.9927 16 Biotinidase Activity + AAAC MS/MS Assay AC Correlation to AAAC only assay AC SLOPE R2 C0 1.05 0.9994 C2 1.12 0.9902 C3 1.11 0.9915 C4 1.10 0.9946 C5DC 0.89 0.9641 C6 0.99 0.9973 C8 1.00 0.9987 C10 1.05 0.9987 C12 1.01 0.9968 C14 1.06 0.9952 C16 1.21 0.9837 C18 1.16 0.9844 MEAN 1.06 0.9912 17 Biotinidase Activity + AAAC MS/MS Assay AAAC within run CV (MRM) AA CV AC CV ALA 5.5% C0 5.8% ARG 8.2% C2 6.0% CIT 8.5% C3 6.5% GLY 22.2% C4 6.0% LEU 8.5% C5DC 13.2% MET 4.1% C6 5.9% ORN 6.0% C8 11.5% OXO-PRO 8.1% C10 6.1% PHE 8.3% C12 5.7% PRO 5.7% C14 4.3% TYR 5.0% C16 4.2% VAL 6.7% C18 7.3% 18 Conclusions Ø Proof of concept for… • Combined Biotinidase activity + AAAC MS/MS assay »Good performance on Biotinidase assay »No compromise in AAAC assay Ø Potential Advantages… • Internal standardization • Assay consolidation Ø We will continue with research… • We are at very early stages… 19 Thank you 20 20.
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