Quantification of ATP7B Protein in Dried Blood Spots by Peptide Immuno-SRM as a Potential Screen for Wilson's Disease

Abstract

Wilson's Disease (WD), a copper transport disorder caused by a genetic defect in the ATP7B gene, has been a long time strong candidate for newborn screening (NBS), since early interventions can give better results by preventing irreversible neurological disability or liver cirrhosis. Several previous pilot studies measuring ceruloplasmin (CP) in infants or children showed that this marker alone was insufficient to meet the universal screening for WD. WD results from mutations that cause absent or markedly diminished levels of ATP7B. Therefore, ATP7B could serve as a marker for the screening of WD, if the protein can be detected from dried blood spots (DBS). This study demonstrates that the immuno-SRM platform can quantify ATP7B in DBS in the picomolar range, and that the assay readily distinguishes affected cases from normal controls (p < 0.0001). The assay precision was <10% CV, and the protein was stable for a week in DBS at room temperature. These promising proof-of-concept data open up the possibility of screening WD in newborns and the potential for a multiplexed assay for screening a variety of congenital disorders using proteins as biomarkers in DBS.

Keywords: ATP7B; DBS; NBS; WD; Wilson’s disease; dried blood spots; immuno-SRM; mass spectrometry; newborn screening; peptide immunoaffinity enrichment.

Figure 1

A, Mass Spectrum of heavy peptide 1056 for ATP7B and B, Tandem mass spectrum of the most abundant parent ion (M+3H). Abundant fragments are selected and optimized for SRM analysis. C, Total ion chromatogram (TIC) and SRM spectra of endogenous (top) and heavy (bottom) peptide 1056 observed in HepG2 cell extract. Chromatographic peaks overlap and SRM patterns are compatible.

Figure 2. Extracted ion chromatograms for ATP7B 1056 peptide after peptide capture in normal PBMC

Top panel is a signature peptide found in the PBMC. Bottom panel is the isotopically labeled internal standard. Chromatographic peaks overlap, and SRM patterns are comparable. Transition labels refer to the precursor charge, fragment ion, fragment m/z, and fragment charge state.

Figure 3. Response curve for ATP7B 1056 peptide

Curves are plotted for the sum of all transitions. The inset plot shows more detail of lower end of the concentration range. Error bars are the standard deviation of three process replicates.

Figure 4. Stability of ATP7B 1056 peptide in normal control DBS at room temperature and −20°C for 0, 3, and 7 days

The data represent the average of three replicates. Dashed and solid lines represent ATP7B concentrations and percent difference, respectively. Error bars are the standard deviation of three process replicates.

Figure 5. Extracted ion chromatograms for ATP7B 1056 peptide after peptide capture in DBS from (A) normal control and (B) WD patient

Top panel is a signature peptide found in DBS. Bottom panel is the isotopically labeled internal standard. Chromatographic peaks overlap and SRM patterns are comparable. Transition labels refer to the precursor charge, fragment ion, fragment m/z, and fragment charge state.

Figure 6. Distribution of the levels of ATP7B in DBS from 13 WD patients and 12 normal controls

The bold black line indicates the median, the inner quartiles are represented by boxes, and the whiskers show 95% of the data.