Related Experiment Video
Updated: Nov 5, 2025
High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry
Published on: April 23, 2019
Development of a multiplex mass spectrometry method for simultaneous quantification of urinary proteins related to
Sarah J D Nauwelaerts1,2, Nancy H C Roosens1, Alfred Bernard2
1Transversal Activities in Applied Genomics, Sciensano, Brussels, Belgium.
Insights
A new method accurately measures urinary proteins, aiding children's respiratory health monitoring. This technique quantifies biomarkers for airway health and kidney function, using urine as a non-invasive sample source.
Area of Science:
- Biochemistry
- Clinical Chemistry
- Pediatric Pulmonology
Background:
- Children's respiratory health is a key concern, with biomarkers like club cell protein (CC16) indicating airway epithelium integrity.
- Urinary biomarkers offer a non-invasive approach for pediatric studies, but require adjustments for confounders like renal function and urinary dilution.
- Osteopontin (OPN) and nuclear factor-kappa B (NF-κB) are also implicated in respiratory health.
Purpose of the Study:
- To develop and validate a simultaneous quantification method for seven key urinary proteins in children.
- To assess the utility of urine as a surrogate source for biomarkers of airway epithelium integrity and renal function.
- To facilitate improved monitoring of children's respiratory health.
Main Methods:
- A multiple reaction monitoring (MRM) method was developed and validated for the relative quantification of seven urinary proteins: CC16, OPN, NF-κB, beta-2-microglobulin (β2M), retinol binding protein 4 (RBP4), myoglobin (MYO), and human serum albumin (HSA).
- Nine proteotypic peptides were selected for quantification.
- The method's validation included assessment of limits of quantification (LOQ), reproducibility, linearity, and comparison with immunoassay data.
Main Results:
- The validated MRM method demonstrated good reproducibility and linearity (r² > 0.98) with LOQ's ranging from 0.3 to 42.8 ng/ml.
- Beta-2-microglobulin (β2M) and retinol binding protein 4 (RBP4) were identified as suitable candidates for adjusting for renal handling and dysfunction.
- All targeted proteins were detected in urine samples, except for MYO and NF-κB.
Conclusions:
- The validated MRM method enables simultaneous quantification of urinary biomarkers for airway epithelium integrity and renal function in children.
- This approach supports the investigation of urine as a reliable surrogate for respiratory health biomarkers in pediatric populations.
- The method provides a valuable tool for understanding the complex factors influencing children's respiratory health.
Abstract:
Respiratory health of children is a health priority. Club cell protein (CC16) is an interesting biomarker of lung diseases and adverse effects towards the airway epithelium integrity. Osteopontin (OPN) and nuclear factor-kappa B (NF-κB) also play a role in respiratory health. The use of urine as biomarker source is useful in studies involving children but necessitates proper adjustment for physiological confounders influencing the urinary excretion, potentially characterized with beta-2-microglobulin (β2M), retinol binding protein 4 (RBP4) or myoglobin (MYO), as well as adjustment for possible renal dysfunction, characterized by human serum albumin (HSA). The simultaneous quantification of all these proteins in urine could facilitate children's health monitoring. A multiple reaction monitoring method (MRM) was developed and validated for the relative quantification of the seven mentioned urinary proteins. A total of nine proteotypic peptides were selected and used for the relative quantification of the seven proteins. The MRM method was completely validated for all proteins and partially for OPN. LOQ's ranged from 0.3 to 42.8 ng/ml, a good reproducibility and a good linearity were obtained across the analytical measurement range (r2 > 0.98). The method yielded varying correlations (r2 of 0.78, 0.71, 0.34 and 0.15 for CC16, β2M, RBP4 and HSA respectively) with available immunoassay data. It also allowed the identification and successful quantification of β2M and RBP4 as a protein candidate for adjustment of renal handling and dysfunction. All proteins were detected in the urine samples except for MYO and NF-κB. Our validated MRM-method is able to simultaneously quantify in urine biomarkers of airway epithelium integrity and biomarkers of variation in renal function and urinary dilution. This will allow to investigate further in future studies if urine can be used as a good surrogate source for biomarkers of airway epithelium integrity, and to understand the complex relationship between cause and effect in children's respiratory health monitoring.
