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