Development of a Reference Measurement Procedure to Certify Urinary Albumin
Ashley S. Beasley, David Bunk, and Karen Phinney
National Institute of Standards and Technology
Analytical Chemistry Division
Urinary excretion of albumin is a major diagnostic and prognostic marker of renal dysfunction and cardiovascular disease; therefore, accurate measurement of urine albumin is vital to clinical diagnosis. Although albumin is one of the most abundant proteins in the urinary proteome, heterogeneity of urinary albumin may reduce the accuracy and validity of current affinity-based methodologies. Therefore, we plan to develop a reference measurement procedure that utilizes isotope dilution-mass spectrometry (ID-MS) and multiple reaction monitoring (MRM) to certify albumin in human urine.
Quantitative ID-MS was performed on the Applied Biosystems API 4000 triple quadrupole mass spectrometer (Foster City, CA) utilizing MRM scanning technique to analyze specific precursor/product MRM transitions. Isotopically-labeled (15N) full-length recombinant human serum albumin (15N-rHSA) was used as the internal standard and spiked into urine samples prior to trypsin digestion. Control HSA (rHSA) was digested with trypsin under several conditions to determine the optimal digestion conditions, protein sequence coverage of trypsin, and optimal MRM transitions for urinary albumin quantitation. The 15N-rHSA internal standard was spiked into various concentrations of rHSA in control urine (NIST SRM 3667) to investigate the linear relationship between peptide concentration and peak intensity of the selected MRM transitions. The peak intensity, retention time, and peak shape of each analyte:internal standard MRM transition pair was analyzed for each standard curve sample. Pooled human urine containing an albumin concentration of 160 mg/L was used to analyze the validity of the MS-based reference measurement procedure for urinary albumin quantitation.