Quantification of urinary albumin by using protein cleavage and LC-MS/MS.
Quantification of urinary albumin by using protein cleavage and LC-MS/MS.
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DOI：
10.1373/clinchem.2008.115543
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发表时间：
2009-06
影响因子：
9.3
通讯作者：
Kumar R
中科院分区：
文献类型：
--
作者：
Seegmiller JC;Barnidge DR;Burns BE;Larson TS;Lieske JC;Kumar R
尿白蛋白排泄是肾脏疾病的敏感诊断和预后标志物。因此，尿白蛋白的测量必须准确、精密。我们开发了一种定量下限 (LOQ) 的完整尿白蛋白的方法。我们使用添加到炭剥离尿液基质中的纯化人血清白蛋白 (HSA) 构建了外部校准曲线。然后，我们将内标、15N 标记的重组 HSA (15NrHSA) 添加到校准品、对照品和患者尿液样本中。将样品还原、烷基化并用胰蛋白酶消化。通过液相色谱-串联质谱（LC-MS/MS）和线性回归分析测定每个样品中白蛋白的浓度，其中白蛋白和15NrHSA中胰蛋白酶肽42LVNEVTEFAK51和526QTALVELVK534的相对丰度面积比参考校准曲线。定量下限为3.13 mg/L，线性动态范围为3.13~200 mg/L。低、中和高对照 (n = 5) 的重复消化物导致肽 42LVNEVTEFAK51 的测定内不精确 CV 为 2.8%–11.0%，肽 526QTALVELVK534 的测定内不精确 CV 为 1.9%–12.3%。连续 10 天 (n = 10) 的对照测定间不精密度导致 42LVNEVTEFAK51 肽的 CV 为 1.5%–14.8%，526QTALVELVK534 肽的 CV 为 6.4%–14.1%。对于 42LVNEVTEFAK51 肽，138 名患者样品的 LC-MS/MS 与免疫比浊法之间的方法比较得出 R2 值为 0.97，回归线为 y = 0.99x + 23.16。尿白蛋白可通过蛋白质裂解 LC-MS/MS 方法使用 15NrHSA 内标进行定量。与市售的免疫比浊测定法相比，该方法在临床相关范围内提供了改进的分析性能。
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). 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. 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. 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.
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影响因子：
4.4
作者：
Hanke, Stefan;Besir, Hueseyin;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
4.4
作者：
Barnidge, DR;Goodmanson, MK;Muddiman, DC
通讯作者：
Muddiman, DC
影响因子：
3.5
作者：
MORTENSEN, HB;MARINELLI, K;PARVING, HH
通讯作者：
PARVING, HH
影响因子：
9.3
作者：
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通讯作者：
Hortin, Glen L.
影响因子：
16.2
作者：
Meigs, JB;D'Agostino, RB;Wilson, PWF
通讯作者：
Wilson, PWF