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

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.

Candidate High-Resolution Mass Spectrometry-Based Reference Method for the Quantification of Procalcitonin in Human Serum Using a Characterized Recombinant Protein as a Primary Calibrator

“…The SI-traceable quantification of Met-PCT [3–116] standard was performed by amino acid analysis (AAA) as described previously. 24 Briefly, the Met-PCT [3–116] content was determined by quantifying phenylalanine, proline, valine, and leucine by ID-LC-MS using a five-point calibration curve after gas-phase hydrolysis in acidic conditions. Conditions of the gas-phase hydrolysis were optimized by carrying out the gas-phase hydrolysis (ELDEX Workstation) at different conditions: 110, 130, or 150 °C for 40 h and 130 °C for 24 and 72 h. In each condition, four processed replicates were performed.…”

“…The calibration, QC materials, and patient samples were processed as described previously. 24 Briefly, 500 μL of serum was subjected to protein denaturation using SDC detergent and precipitated using acetonitrile. Next, the supernatant was diluted and purified on a C18 solid-phase extraction (SPE) cartridge (HLB C18, Waters).…”

“…SALESSPADPATLSEDEAR (noted SAL) and FHTFPQTAIGVGAPGK (noted FHT) proteotypic peptides have been previously selected for PCT quantification. 24 Two transitions per peptide were selected, one used as peptide quantifier and another as peptide qualifier (see Table S3 , Supporting Information). Raw data were processed with Xcalibur software v4.1 (Thermo Scientific).…”

Letter to the Editor regarding “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”

“…In their article in this journal, Huynh et al describe a method for PCT quantification by isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) [3]. The enrichment of PCT out of the complex serum matrix was achieved by two steps of solid-phase extraction (SPE), initially for enrichment of intact PCT from serum and subsequently for capture of PCT peptides generated by trypsin digestion.…”

Response to Letter to the Editor regarding “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”

“…Huu-Hien Huynh Dear Editor, In this issue, Tölke et al responded to our recently published paper describing an antibody-free method for procalcitonin (PCT) quantification by isotope-dilution coupled to liquid chromatography and tandem mass spectrometry (ID-LC-MS/MS) [1]. This group proposes a MS-based quantification strategy that relies on polyclonal antibodybased enrichment for PCT affinity purification at the protein level [yet unpublished].…”