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Letters to the Editor |
1 Department of Laboratory, Medicine and Pathology
2 Nephrology Research Unit Department of Internal Medicine
3 Department of Biochemistry, and Molecular Biology, Mayo Clinic, Rochester, MN
aAddress correspondence to this author at: 200 First St SW, Rochester, MN 55905. Fax 507-284-9758; e-mail singh.ravinder{at}mayo.edu.
To the Editor:
Recent studies suggest that microalbuminuria confers increased cardiovascular risk, even in nondiabetic and nonhypertensive persons (1)(2). Clinically, the testing of microalbuminuria uses immunochemical methods. Controversy exists, however, regarding the accuracy of immunochemical and chromatographic methods for quantifying urine albumin (3). We earlier reported on liquid chromatography-mass spectrometry (LC-MS) for measurement of urinary albumin with bovine serum albumin (BSA) as an internal standard (4). We now report the preparation of 15N-labeled albumin as an internal standard and the validation of its use in an LC-MS assay for quantifying low concentrations of albumin in the urine of patients.
The Pichia pastoris strain GS115/His+Muts (Invitrogen) was used to synthesize 15N-labeled human serum albumin (HSA) [see details in the Data Supplement that accompanies the online version of this Letter at http://www.clinchem.org/content/vol53/issue3]. A Q-TOF PremierTM mass spectrometer (Waters/Micromass) was used to characterize the 15N-labeled HSA and study its fragmentation. Loo et al. (5) previously reported the generation of source-induced fragmentation of intact proteins in an electrospray ionization source of a mass spectrometer. They demonstrated production of b-series fragments consistent with the sequence of albumin from 10 different species. We confirmed these results with HSA, BSA, and 15N-labeled HSA on the Q-TOF Premier by raising the cone voltage from 40 to 80 volts. The highly charged molecular ions virtually disappear and are replaced by smaller charged fragment ions with charge states ranging from +2 to +6 (Fig. 1
, A-C). These fragments are consistent with b-series fragment ions originating from the N-terminus of the HSA molecule.
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To certify the purity of the 15N-labeled HSA, we compared the source-induced-fragmentation spectrum (specifically the b24+4 ions) of labeled HSA with the coinciding ion obtained from commercial HSA (Fig. 1
, D and E). For unlabeled HSA, the lowest expected mass for the isotope cluster of this ion was observed at the expected mass of m/z = 684.6152. For the labeled material, the signal at this mass vanished and a signal was observed at m/z = 693.3276, indicating virtually complete 15N-labeling of the 35 nitrogens in the b24 fragment. Calculations indicated that each individual nitrogen in the protein was 98.95% 15N-labeled.
Because of low throughput, the Q-TOF mass spectrometer is not practical for routine clinical testing. Therefore, for routine clinical use, we validated the method using an API 5000TM triple quadrupole mass spectrometer (Applied Biosystems). Since tuning values of the API 5000 differ from those of the Q-TOF, the method was further optimized for the API 5000. The N-terminal fragment ions corresponding to unlabeled b244+ (m/z = 685.1) and b243+(m/z = 693.6) were used for quantification of HSA using 15N-HSA as an internal standard: 15N35-b244+(m/z = 913.2) and 15N35-b243+ (m/z = 924.1). The chromatographic conditions were the same as described earlier (4).
Our precision studies yielded intraassay CVs (n = 20) of 12.6% (17 mg/L), 10.1% (70 mg/L), and 4.0% (210 mg/L), and the interassay CVs (n = 10) were 12.2% (19 mg/L), 11.0% (80 mg/L), and 7.1% (230 mg/L). By running 20 replicate blanks we determined that the lowest analyte concentration indistinguishable from the blank was 2.5 mg/L. By running 20 replicates of a 5 mg/L sample, we established the limit of detection, defined as the lowest analyte concentration that can be distinguished from blank with >95% certainty, as 4.84 mg/L. The limit of quantification/functional sensitivity was set at 10.5 mg/L, corresponding to the lowest control patient samples with an interassay CV <20%.
Multiple calibration curves ranging from 4 to 625 mg/L were linear and were reproducible with the following linear regression equation: y = 0.01x + 0.20 (r2 = 0.999).
The linearity of the assay was assessed by extracting 5 patient samples at the following serial dilutions: undiluted, 1:2, 1:4, and 1:8. The expected value for each sample was calculated based on the result obtained for the undiluted sample. The percentages of the expected results were 87%–121% (mean 98%) for urine specimens containing 80–207 mg/L albumin. In additional experiments, HSA was added at 3 concentrations (40, 80, and 170 mg/L) into patient samples (n = 5) and assayed in single determinations. For individual samples, calculated recoveries ranged from 93% to 117% (mean 105%). The comparison shows that the LC-MS method using 15N-HSA is largely in agreement with the Hitachi method (y = 0.96x + 7.25, R2 = 0.99), similar to when BSA was employed as the internal standard. In conclusion, we have developed a sensitive and specific LC-MS assay for detection and measurement of urinary albumin that employs 15N-HSA as a unique and novel internal standard.
References
The following articles in journals at HighWire Press have cited this article:
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  J. C. Seegmiller, D. R. Barnidge, B. E. Burns, T. S. Larson, J. C. Lieske, and R. Kumar Quantification of Urinary Albumin by Using Protein Cleavage and LC-MS/MS Clin. Chem., June 1, 2009; 55(6): 1100 - 1107. [Abstract] [Full Text] [PDF]
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E. Kuhn, T. Addona, H. Keshishian, M. Burgess, D.R. Mani, R. T. Lee, M. S. Sabatine, R. E. Gerszten, and S. A. Carr Developing Multiplexed Assays for Troponin I and Interleukin-33 in Plasma by Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Clin. Chem., June 1, 2009; 55(6): 1108 - 1117. [Abstract] [Full Text] [PDF]
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W. G. Miller, D. E. Bruns, G. L. Hortin, S. Sandberg, K. M. Aakre, M. J. McQueen, Y. Itoh, J. C. Lieske, D. W. Seccombe, G. Jones, et al. Current Issues in Measurement and Reporting of Urinary Albumin Excretion Clin. Chem., January 1, 2009; 55(1): 24 - 38. [Abstract] [Full Text] [PDF]
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A. Shaikh, J. C. Seegmiller, T. M. Borland, B. E. Burns, P. M. Ladwig, R. J. Singh, R. Kumar, T. S. Larson, and J. C. Lieske Comparison between Immunoturbidimetry, Size-Exclusion Chromatography, and LC-MS to Quantify Urinary Albumin Clin. Chem., September 1, 2008; 54(9): 1504 - 1510. [Abstract] [Full Text] [PDF]
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