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 employ tandem mass spectrometry (MS/MS) to characterize the various molecular forms of urinary albumin and isotope dilution-mass spectrometry (ID-MS) to quantify albumin in human urine.
Prior to characterization of the urinary albumin species, a non-antibody-based method, including off-line liquid chromatography (LC) and ultrafiltration, was utilized to isolate the albumin species from control and disease urine. In addition to enriching the albumin sub-proteome, the reproducible off-line LC and ultrafiltration methods were also used to fractionate the albumin species present in the urine samples. Following proteolytic digestion of the enriched albumin species, qualitative characterization was accomplished using on-line LC tandem MS with a linear ion trap mass spectrometer (LTQ-XL, ThermoFisher, San Jose, CA) operated in the data-dependent acquisition mode. Quantitative ID-MS was performed on the Applied Biosystems API 5000 triple quadrupole mass spectrometer (Foster City, CA) utilizing the multiple reaction monitoring (MRM) scanning technique. The MultiQuantTM software (Foster City, CA) was used to evaluate the peak intensity, retention time, and peak shape of each MRM transition. Isotopically-labeled (15N) full-length recombinant human albumin (15Nr-HSA) was used as the internal standard and spiked into control and disease urine prior to trypsin digestion.
Human serum albumin standard (std-HSA) was digested with trypsin under several conditions and analyzed via LC-MS/MS on the LTQ-XL to determine the optimal digestion conditions and evaluate the protein sequence coverage of trypsin. The std-HSA tryptic fragments were also analyzed on the API 5000 triple quadrupole to determine optimal MRM transitions for albumin quantitation. The effects of the urine matrix on albumin characterization and quantitation were also measured by analyzing control urine samples (NIST SRM 3667) spiked with std-HSA at varying concentrations (25 mg/L, 150 mg/L, and 400 mg/L) via MRM and tandem MS. In addition to the matrix effects, the std-HSA was also used to investigate albumin enrichment efficiency, digestion reproducibility, and tandem MS repeatability. Urinary albumin concentrations in control and disease urine were determined from the differential expression of target albumin peptides and corresponding 15Nr-HSA standard. |
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