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Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

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Abstract

The quantification of low abundant proteins in complex matrices by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains challenging. A measurement procedure based on optimized antibody-free sample preparation and isotope dilution coupled to LC-MS/MS was developed to quantify procalcitonin (PCT) in human serum at sub-microgram per liter level. A combination of sodium deoxycholate-assisted protein precipitation with acetonitrile, solid-phase extraction, and trypsin digestion assisted with Tween-20 enhanced the method sensitivity. Linearity was established through peptide-based calibration curves in the serum matrix (0.092–5.222 μg/L of PCT) with a good linear fit (R2 ≥ 0.999). Quality control materials spiked with known amounts of protein-based standards were used to evaluate the method’s accuracy. The bias ranged from −2.6 to +4.3%, and the intra-day and inter-day coefficients of variations (CVs) were below 2.2% for peptide-based quality controls. A well-characterized correction factor was determined and applied to compensate for digestion incompleteness and material loss before the internal standards spike. Results with metrological traceability to the SI units were established using standard peptide of well-characterized purity determined by peptide impurity corrected amino acid analysis. The validated method enables accurate quantification of PCT in human serum at a limit of quantification down to 0.245 μg/L (bias −1.9%, precision 9.1%). The method was successfully applied to serum samples obtained from patients with sepsis. Interestingly, the PCT concentration reported implementing the isotope dilution LC-MS/MS method was twofold lower than the concentration provided by an immunoassay.

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Abbreviations

AAA:

Amino acid analysis

ACN:

Acetonitrile

CRM:

Certified reference material

CV:

Coefficient of variation

DMSO:

Dimethyl sulfoxide

ID-LC-MS/MS:

Isotope dilution liquid chromatography tandem mass spectrometry

HLB:

Hydrophilic-lipophilic balance

HRMS:

High-resolution mass spectrometry

LOQ:

Limit of quantification

PCT:

Procalcitonin

PICAA:

Peptide impurity corrected amino acid strategy

PRM:

Parallel reaction monitoring

QC:

Quality control

SDC:

Sodium deoxycholate

SIL:

Stable isotope labeled

SPE:

Solid-phase extraction

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Acknowledgments

We would like to thank Dr. Qinde Liu and Dr. Yizhao Chen from Health Sciences Authority (HAS) of Singapore for scientific discussions.

Funding

This work was supported by the European Metrology Programme for Innovation and Research (EMPIR) joint research projects [15HLT07] “AntiMicroResist” and [18HLT03] “SEPTIMET” which have received funding from the EMPIR programme co-financed by the Participating States and the European Union’s Horizon 2020 research and innovation programme. Huu-Hien Huynh and Maxence Derbez-Morin were supported by a CIFRE scholarship provided by ANRT (Association Nationale de la Recherche et de la Technologie). Conseil Régional d’Île-de-France subsidized SMBP mass spectrometry equipment (Sesame 2010 No. 10022268).

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Authors and Affiliations

  1. Biomedical and Organic Chemistry Department, Laboratoire National de Métrologie et d’Essais (LNE), Paris, France

    Huu-Hien Huynh, Amandine Bœuf, Maxence Derbez-Morin, Béatrice Lalere & Vincent Delatour

  2. Biological Mass Spectrometry and Proteomics, SMBP, ESPCI Paris, Université PSL, CNRS FRE2032, Paris, France

    Huu-Hien Huynh & Joëlle Vinh

  3. Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, Gif-sur-Yvette, France

    Maxence Derbez-Morin

  4. Laboratoire de Biochimie et Hormonologie, CHU Montpellier, Université Montpellier 1, Montpellier, France

    Anne-Marie Dupuy

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Huynh, HH., Bœuf, A., Derbez-Morin, M. et al. Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration. Anal Bioanal Chem 413, 4707–4725 (2021). https://doi.org/10.1007/s00216-021-03361-0

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