Kidney injury is
a complication frequently encountered in hospitalized
patients. Early detection of kidney injury prior to loss of renal
function is an unmet clinical need that should be targeted by a protein-based
biomarker panel. In this study, we aim to quantitate urinary kidney
injury biomarkers at the picomolar to nanomolar level by liquid chromatography
coupled to tandem mass spectrometry in multiple reaction monitoring
mode (LC-MRM-MS). Proteins were immunocaptured from urinary samples,
denatured, reduced, alkylated, and digested into peptides before LC-MRM-MS
analysis. Stable-isotope-labeled peptides functioned as internal standards,
and biomarker concentrations were attained by an external calibration
strategy. The method was evaluated for selectivity, carryover, matrix
effects, linearity, and imprecision. The LC-MRM-MS method enabled
the quantitation of KIM-1, NGAL, TIMP2, IGFBP7, CXCL9, nephrin, and
SLC22A2 and the detection of TGF-β1, cubilin, and uromodulin.
Two to three peptides were included per protein, and three transitions
were monitored per peptide for analytical selectivity. The analytical
carryover was <1%, and minimal urine matrix effects were observed
by combining immunocapture and targeted LC-MRM-MS analysis. The average
total CV of all quantifier peptides was 26%. The linear measurement
range was determined per measurand and found to be 0.05–30
nmol/L. The targeted MS-based method enables the multiplex quantitation
of low-abundance urinary kidney injury biomarkers for future clinical
evaluation.