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Abstract: SA-PO1113

Genotyping Apolipoprotein L1 (ApoL1) from Dry Plasma with Proteomics

Session Information

Category: Pathology and Lab Medicine

  • 1502 Pathology and Lab Medicine: Clinical

Authors

  • Shuford, Christopher M., Laboratory Corporation of America, Burlington, North Carolina, United States
  • Bradley, Meghan N., Laboratory Corporation of America, Burlington, North Carolina, United States
  • Holland, Patricia L., Laboratory Corporation of America, Burlington, North Carolina, United States
  • Levandoski, Michael, Laboratory Corporation of America, Burlington, North Carolina, United States
  • Ennis, Jennifer L., Laboratory Corporation of America, Burlington, North Carolina, United States
  • Asplin, John R., Laboratory Corporation of America, Burlington, North Carolina, United States
  • Grant, Russell P., Laboratory Corporation of America, Burlington, North Carolina, United States
Background

ApoL1 has two genetic risk variants (G1 & G2) associated with increased risk of nondiabetic CKD and kidney transplant failure. As these risk variants occur almost exclusively in African Americans, genetic testing may improve both monitoring and outcomes of CKD as well as allocation of donor organs in this population. To this end, we developed a bottom-up proteomic assay to identify genetic variants of ApoL1 from both liquid plasma samples collected by venipuncture as well as from dry plasma samples produced on a blood plasma separation device that can be collected in-home via a finger prick.

Methods

Both 20 µL of plasma or ~95 mm2 punch of dried plasma were processed using a common workflow. Samples were denatured in DTT and deoxycholate for 30min at 56°C, then digested with 800µg of trypsin for 30min at 37°C (pH 8) after addition of labeled internal standards. Digestion was quenched and the deoxycholate precipitated by the addition of formic acid and the clarified supernatant analyzed by liquid chromatography coupled with tandem mass spectrometry. The three ApoL1 protein variants (WT, G1, G2) were uniquely detected by monitoring for the specific surrogate peptides containing the point mutations, which were produced by the trypsin digestion. Two surrogate peptides common among all protein variants were also monitored for quality control of sample processing.

Results

Reproducibility of the proteomic assay was demonstrated over 20 assay runs by analysis of dry and liquid specimens from 3 heterozygous individuals (WT/G1, WT/G2, and G1/G2). Dry plasma from the same heterozygous genotypes were observed to be stable for up to 4 weeks stored at ambient (22°C) and elevated temperature (37°C). Finally, perfect agreement with a Sanger Sequencing assay was observed in matched liquid and dry plasma specimens in a cohort of 209 African American donors, which consisted of 82 WT/WT, 64 WT/G1, 38 WT/G2, 10 G1/G1, 10 G1/G2, and 5 G2/G2 individuals.

Conclusion

This proteomic assay provides the feasibility for ApoL1 genotyping using dry plasma which can be collected in-home via finger prick using blood plasma separation devices. Such testing should allow for more widespread and cost-effective genotyping of this high risk population.