Analysis of the Low-Abundance Plasma Biomarker Klotho in Less than Four Hours

Significance of the Topic

Precise quantification of low-abundance protein biomarkers in plasma is crucial for aging research and clinical diagnostics. Klotho, a transmembrane β-glucuronidase linked to aging and kidney disease, circulates at low levels in biological matrices. Traditional workflows for protein enrichment and digestion are time-consuming and can limit throughput and reproducibility.

Objectives and Study Overview

This study aimed to develop and demonstrate a streamlined LC-MS/MS method for rapid quantitation of plasma Klotho. By integrating immunoaffinity capture and digestion in a single-step workflow using the SMART Digest ImmunoAffinity kit, the goal was to achieve high sensitivity and reproducibility in less than four hours.

Methodology and Instrumentation

The workflow combined antibody biotinylation, target capture, on-bead digestion, and peptide analysis.

• Antibody biotinylation with NHS-biotin at room temperature for 2 hours
• Immunoaffinity capture using streptavidin-coated SMART Digest IA beads: 2-hour incubation at ambient temperature
• Sequential wash steps to remove non-specific matrix components
• On-bead digestion at 70 °C for 90 minutes using immobilized trypsin in denaturing conditions
• Peptide separation on a Thermo Scientific Accucore C18 column (2.1×50 mm, 2.6 μm) with a water/acetonitrile/formic acid gradient
• Detection with a Thermo Scientific Velos Pro ion trap mass spectrometer in positive mode

Main Results and Discussion

The method achieved a lower limit of quantitation (LLOQ) of 1.58 ng/mL in mouse plasma for two signature peptides (FSISWAR and LQDAYGGWANR). Calibration curves over 1.58–500 ng/mL exhibited strong linearity (R2 > 0.97). Precision at LLOQ demonstrated coefficients of variation of 6.3% and 12.5% for the two peptides, with overall CVs below 17% across the calibration range. Total sample preparation and analysis time was reduced to approximately four hours compared to 15–24 hours in conventional workflows.

Benefits and Practical Applications

• Rapid and integrated immunoenrichment and digestion reduces hands-on time and potential errors
• High sensitivity and reproducibility suitable for low-abundance biomarker quantitation
• Compatibility with automation supports high-throughput laboratory environments
• Versatile approach applicable to other protein biomarkers and isoforms in complex biological matrices

Future Trends and Potential Applications

Advances are expected in multiplexed immunoaffinity–MS workflows, increased automation, and adaptation to microfluidic platforms for further throughput gains. Integration with novel antibody formats, high-resolution mass spectrometry, and data-independent acquisition may expand sensitivity and enable broader biomarker panels in clinical research and precision medicine.

Conclusion

The presented SMART Digest IA workflow provides a fast, simple, and sensitive LC-MS/MS method for quantifying low-level plasma Klotho. By co-immobilizing capture reagents and trypsin on magnetic beads, the protocol achieves reliable results in under four hours, offering a robust tool for biomarker studies in aging and kidney disease.

References

• Rodriguez T. Identifying significant biological markers in Klotho gene variants across wide ranging taxonomy. Journal of Molecular Biology Research. 2015;5(1):11.
• Rotondi S, Pasquali M, Tartablione L, et al. Soluble α-Klotho serum levels in chronic kidney disease. International Journal of Endocrinology. 2015;872193.
• Massó A, Sánchez A, Gimenez-Llort L, et al. Secreted and Transmembrane α-Klotho Isoforms have different spatiotemporal profiles in the brain during aging and Alzheimer’s disease progression. PLoS ONE. 2015;10(11):e0143623.
• Lim K, Groen A, Molostvov G, et al. α-Klotho expression in human tissues. Journal of Clinical Endocrinology and Metabolism. 2015;100(10):e1308-1318.