Received on August 5, 2008
Accepted on December 23, 2008

Proteomics and Protein Markers

Quantification of Urinary Albumin by Using Protein Cleavage and LC-MS/MS

¹ Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN
² Department of Laboratory Medicine and Pathology, Department of Internal Medicine, and Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN
³ Department of Laboratory Medicine and Pathology, and Department of Internal Medicine, Mayo Clinic, Rochester, MN
⁴ Department of Internal Medicine, Division of Nephrology and Hypertension, Division of Endocrinology, Diabetes, Metabolism and Nutrition, and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN

^* To whom correspondence should be addressed. E-mail: rkumar{at}mayo.edu.

BACKGROUND: Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We investigated a method to quantify intact urinary albumin with a low detection limit.

METHODS: We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, ¹⁵N-labeled recombinant HSA (¹⁵NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides ⁴²LVNEVTEFAK⁵¹ and ⁵²⁶QTALVELVK⁵³⁴ from albumin and ¹⁵NrHSA were referenced to the calibration curve.

RESULTS: The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13–200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%–11.0% for the peptide ⁴²LVNEVTEFAK⁵¹, and 1.9%–12.3% for the ⁵²⁶QTALVELVK⁵³⁴ peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%–14.8% for the ⁴²LVNEVTEFAK⁵¹ peptide, and 6.4%–14.1% for the ⁵²⁶QTALVELVK⁵³⁴ peptide. For the ⁴²LVNEVTEFAK⁵¹ peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R² value of 0.97 and a regression line of y = 0.99x + 23.16.

CONCLUSIONS: Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a ¹⁵NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.