Quantification of hemoglobin A2 by tandem mass spectrometry
Quantification of hemoglobin A2 by tandem mass spectrometry
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DOI：
10.1373/clinchem.2007.088682
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发表时间：
2007-08-01
影响因子：
9.3
通讯作者：
Dalton, Neil
中科院分区：
文献类型：
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作者：
Daniel, Yvonne A.;Turner, Charles;Dalton, Neil
背景资料：在多反应监测（MRM）模式下使用电喷雾串联质谱（MSMS）进行基于肽的全血分析，能够快速检测和序列确认具有临床意义的血红蛋白（Hb）变体。我们采用了类似的定量方法来测量δ：β-珠蛋白肽的比例，作为Hb（2）的潜在替代标记物，Hb（2）是一种用于人群筛查β-地中海贫血特征的生物标记物。方法：我们研究了163例HbA正常的血液样本，105例HbA升高的血液样本，43例δ链变异体，8例Hb Lepore。全部通过HPLC检测。还将样品与胰蛋白酶在37 ° C下孵育30分钟，用于流动注射分析的MSMS。获取δ-（T2、T3和T14）和β-（T2、T3和T13）珠蛋白胰蛋白酶肽的MRM 1分钟，并计算δ：β肽比率。我们使用HPLC和MSMS分析了26对全血和干血斑样品在储存1、8和29天后的含量。结果：δ：β肽比值的批内和批间不精密度值（CV）分别< 6.1%和<8.4%。消化物在10 ℃下稳定6天。MSMS δ：β-珠蛋白肽比值和HPLC HbA（2）之间的显著相关性（P < 0.0001）允许区分HbA（2）浓度升高和参考区间内的浓度，并识别Hb Lepore。这种分化是可重复的MSMS，但不是由HPLC，血斑样本已被储存1 month.Conclusion：本研究验证了定量δ：β-珠蛋白肽比作为替代标记的HbA（2），并证明了潜在的快速肽为基础的MSMS的多重，高通量蛋白质生物标志物的表征和定量。(c)2007年美国临床化学协会。
Background: Peptide-based analysis of whole blood using electrospray tandem mass spectrometry (MSMS) in multiple reaction m onitoring (MRM) mode enables rapid detection and sequence confirmation of clinically significant hemoglobin (Hb) variants. We applied a similar, quantitative approach to the measurement of delta:beta-globin peptide ratios as potential surrogate markers of Hb(2), a biomarker used in population screening for beta-thalassemia trait.Methods: We studied 163 blood samples with normal HbA(2), (%), 105 with increased HbA(2), 43 with delta-chain variants, and 8 with Hb Lepore. All were tested by HPLC. The samples were also incubated with trypsin for 30 min at 37 degrees C for MSMS with flow injection analysis. MRMs for the delta- (T2, T3, and T14) and beta- (T2, T3, and T13) globin tryptic peptides were acquired for 1 min, and delta:beta peptide ratios were calculated. We used HPLC and MSMS to analyze 26 paired whole blood and dried blood spot samples after storage for 1, 8, and 29 days.Results:.Within- and between-assay imprecision values (CVs) were < 6.1% and < 8.4%, respectively, for the delta:beta peptide ratios. Digests were stable at 10 degrees C for 6 days. Significant correlations (P < 0.0001) between MSMS delta:beta-globin peptide ratios and HPLC HbA(2) allowed differentiation between increased HbA(2) concentrations and concentrations within the reference interval and identification of Hb Lepore. This differentiation was repeatable by MSMS, but not by HPLC, after blood spot samples had been stored for 1 month.Conclusion: This study validates the quantitative delta:beta-globin peptide ratio as a surrogate marker of HbA(2) and demonstrates the potential of rapid peptide-based MSMS for multiplexed, high-throughput protein biomarker characterization and quantification. (c) 2007 American Association for Clinical Chemistry.