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Abstract: SA-PO277

Targeted Mass Spectrometry to Detect and Quantify Circulating α-Klotho Isoforms

Session Information

Category: Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)

  • 2000 Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)

Authors

  • Halim, Arvin, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Narayanan, Gayatri, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Moe, Sharon M., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Doud, Emma H., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Mosley, Amber, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Lim, Kenneth, Indiana University School of Medicine, Indianapolis, Indiana, United States
Background

Progress in α-Klotho research has been stunted by the lack of reliable assays that can discriminate between various α-Klotho isoforms which potentially exert differential physiological roles. We designed a novel targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to detect and quantify the known circulating α-Klotho isoforms: soluble full-length Klotho (sFL-K), secreted KL1 (secKL1), soluble KL1 (sKL1) and soluble KL2 (sKL2).

Methods

We developed a parallel reaction monitoring-based (PRM) LC-MS/MS method that exploits unique peptide sequences present at the α2/β-cut site and the C-terminus of secKL1. During assay design, we tested enzymatic digestions of recombinant human sFL-K and secKL1 with commercially available proteases: trypsin, Lys-C, Asp-N, or chymotrypsin. After determining that chymotrypsin successfully created unique peptides covering the various α-Klotho isoforms, 11 synthetic peptides were used to optimize LC-MS/MS parameters and to determine limit of detection and quantification (LOD/LOQ) in solvent matrix. These peptides included chymotrypsin missed cleavages and deamidated Asn residues which were observed in our preliminary studies. We then utilized recombinant sFL-K and secKL1 proteins spiked into Top14 depleted plasma for further optimization and assessment of the assay.

Results

Only chymotrypsin digestion produced peptide signatures that cover the α2/β-cut site to differentiate between sKL1, sKL2 and sFL-K and the unique secKL1 C-terminus to identify secKL1 from sFL-K and sKL1. The LOD/LOQ for synthetic peptide signatures in solvent matrix ranged from 0.02/0.06 to 1.3/3.8 fmol. Synthetic peptides with an intact α2/β-cut site resulted in multiple peaks, most likely due to the high number of prolines in the peptide which can create distinct conformers that were resolved with application of a 60°C column heater. Deamidated peptides were detectable in depleted plasma matrix spiked-in with recombinant sFL-K and secKL1 proteins. sFL-K and sKL1 were detectable in human serum samples.

Conclusion

This is the first α-Klotho assay that can specifically detect and quantify different isoforms of circulating α-Klotho and overcome limitations of antibody-based methods. Further optimization for robustness, reproducibility as well as validation studies are required to assess α-Klotho isoforms in other sample types.

Funding

  • Other NIH Support