Background
LC-MS/MS has enabled the translation of many novel biomarkers to the clinical laboratory, but its potential for measurement of urinary proteins is still unexplored. In this study we examined the correlation and agreement between immunoassay and LC-MS/MS in the quantitation of kidney injury biomarkers and evaluated the application of technical LC-MS/MS meta-data assessment to ensure test result validity.
Methods
NGAL, IGFBP7, TIMP2, and KIM-1 were quantified in 345 urine samples with one multiplex lab-developed test that combines immunocapture with mass spectrometry read-out and 4 singleplex sandwich-type immunoassays. Assay performance and imprecision were monitored by 2 urine-based quality controls. Ion ratios, signal intensity, and retention time were monitored over all study samples.
Results
The LC-MS/MS retention time drift was ≤1.2%, ion ratios were within 20% of the target values at...
Show moreBackground
LC-MS/MS has enabled the translation of many novel biomarkers to the clinical laboratory, but its potential for measurement of urinary proteins is still unexplored. In this study we examined the correlation and agreement between immunoassay and LC-MS/MS in the quantitation of kidney injury biomarkers and evaluated the application of technical LC-MS/MS meta-data assessment to ensure test result validity.
Methods
NGAL, IGFBP7, TIMP2, and KIM-1 were quantified in 345 urine samples with one multiplex lab-developed test that combines immunocapture with mass spectrometry read-out and 4 singleplex sandwich-type immunoassays. Assay performance and imprecision were monitored by 2 urine-based quality controls. Ion ratios, signal intensity, and retention time were monitored over all study samples.
Results
The LC-MS/MS retention time drift was ≤1.2%, ion ratios were within 20% of the target values at concentrations of >100 pmol/L, and peptides originating from the same protein were in agreement (slopes between 1.03 and 1.41). The interassay CV was between 9.3% and 19.1% for LC-MS/MS analysis and between 4.2% and 10.9% for immunoassay. Direct LC-MS/MS analysis was correlated with immunoassay in the quantitation of NGAL (r = 0.93; range: 0.01–37 nmol/L), IGFBP7 (r = 0.80; range: 0.01–2.6 nmol/L), TIMP2 (r = 0.85; range: 0.01–6.3 nmol/L), and KIM-1 (r = 0.70; range 0.01–0.4 nmol/L), but the analytical methodologies differed in measurands and calibration strategies.
Conclusions
LC-MS/MS is explored as a next-generation technology for multiplex urinary protein measurement. It has great potential to overcome nonselectivity and lack of standardization because of its capability of directly measuring well-defined molecular proteins.
Show less
