Development of a multiplex mass spectrometry method for simultaneous quantification of urinary proteins related to 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.

Assessment of the Feasibility of a Future Integrated Larger-Scale Epidemiological Study to Evaluate Health Risks of Air Pollution Episodes in Children

“…Indeed, from previous studies and based on our field study setup, we found that, for the monitoring of protein biomarkers, urine was found to be easily collected and stored. Using an MRM method, previously validated [ 19 ] and applied in a similar study [ 23 ], allowed the simultaneous quantification of U-CC16, a biomarker of lung injury, permeability and respiratory diseases, as well as U-β2M and U-RBP4, two potential adjusters for variations in diuresis and renal handling of urinary proteins. For the genotyping and epigenetic methylation biomarkers, previous studies confirmed that saliva is a good non-invasive source [ 57 , 58 , 59 ], with DNA of such high quality that it could be used as an alternative to blood DNA in epidemiological studies [ 48 , 60 , 61 ].…”

“…Based on our field study setup, we found that, for the monitoring of protein biomarkers, urine could be analyzed using a validated MRM method [ 19 ]. The use of only 500 µL of urine was more than sufficient for performing a trypsin digest and to deliver a qualitative protein sample for analysis.…”

“…However, prior knowledge of these target proteins is needed to include them in the method. Additionally, sometimes technical or biological challenges, inherent to a candidate protein, can lead to unsuccessful measurements [ 19 ].…”

“…Relevant proteins were investigated using urine samples collected during the different phases of the field study by performing a trypsin digest on 500 µL of urine sample. This was followed by quantification with the MRM method, a previously developed and validated high-throughput method based on a mass spectrometry protocol [ 19 ]. It allowed for the simultaneous relative quantification of U-CC16, U-β2M and U-RBP4.…”

“…Recently, as an alternative to serum CC16, urinary CC16 (U-CC16) was measured using a validated multiple reaction monitoring (MRM) method. This allowed the high-throughput, and hence cost-effective, relative quantification of U-CC16, urinary beta-2-microglobulin (U-β2M) and urinary retinol binding protein 4 (U-RBP4) [ 19 ]. The latter are two potential adjusters for the variations in diuresis and renal handling of proteins.…”