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

Abstract

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 have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). 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.

Comparison of Urinary Albumin Quantification by Immunoturbidimetry, Competitive Immunoassay, and Protein-Cleavage Liquid Chromatography–Tandem Mass Spectrometry

“…Furthermore, it has recently been proposed that assays for this clinically important analyte should be standardized by using a reference material and method (1 ). Therefore, in the current study 3 common clinical tests for urine albumin were performed in 2 separate clinical laboratories, and the results were compared to results from a candidate reference LC-MS/MS method previously developed by the Mayo Clinic Department of Laboratory Medicine (6 ). This approach is presumed to detect all albumin species in the sample and avoids potential issues related to epitope recognition by specific antibodies and/or matrix effects.…”

Quantification of growth hormone in serum by isotope dilution mass spectrometry

“…However, in spite of earlier model experiments to extend the scope of the technique to proteins [7] it was not until recently that quantification by IDMS of protein markers in body fluids for diagnostic purposes has been reported [8][9][10][11][12][13]. Measurement of GH by MS has been discussed previously, namely in a feasibility study [14], a discussion of MS as fast alternative method regarding the time needed for development of antibody based assays [15] and with an investigation about optimal conditions for proteolysis of serum samples [16].…”

Availability of urinary albumin measurement in Southern Brazilian Laboratories

“…Surprisingly, it was only recently that a candidate liquid chromatography-mass spectrometry (LC-MS/ MS) was developed as a reference measurement procedure to assess UA 22 . Another reference measurement procedure has been developed in the Mayo Clinic Renal Function Laboratory, using trypsin digestion of whole urine followed by LC-MS/MS 23 . This initiative will allow for the standardization and harmonization of UA measurements.…”