Berna Michael J, Zhen Yuejun, Watson David E, Hale John E, Ackermann Bradley L
Lilly Research Laboratories, Eli Lilly and Company, Greenfield, Indiana 46140, USA.
Anal Chem. 2007 Jun 1;79(11):4199-205. doi: 10.1021/ac070051f. Epub 2007 Apr 21.
Myosin light chain 1 (Myl3) is a 23-kDa isoform of one of the subunits of myosin, a protein involved in muscle contraction. Myl3 is presently being studied as a biomarker of cardiac necrosis to predict drug-induced cardiotoxicity, and in the work presented here, an LC/MS/MS assay was developed and validated to measure Myl3 in rat serum. The key steps in this approach involved immunoaffinity purification of Myl3 from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified using a synthetic peptide standard and a corresponding stable isotope-labeled internal standard, and the results were stoichiometrically converted to Myl3 serum concentrations. Myl3 concentrations were corrected for peptide recovery following immunoprecipitation and digestion (85%) and showed excellent agreement with synthetic peptide standards. Both the synthetic peptide and His-Myl3 protein were used to evaluate assay accuracy (% RE) and precision (% CV), which were measured on each of 3 days. The synthetic peptide was evaluated over the range of 0.073-7.16 nM, while Myl3 protein QC samples prepared in rat serum were evaluated over the range of 0.13-6.62 nM. To prepare control matrix, endogenous Myl3 was immunodepleted from pooled rat serum. Peptide interday accuracy and precision did not exceed 7.6 and 11.1%, and Myl3 interday accuracy and precision did not exceed 12.9 and 13.2%, respectively. Data are presented from the application of this assay to establish a time course in which rats demonstrated a marked increase in Myl3 serum concentrations following administration of isoproterenol, a beta-adrenergic receptor agonist known to induce cardiac injury. This assay is an example of a larger effort in our laboratory to use LC/MS/MS in conjunction with immunoaffinity techniques to evaluate candidate biomarkers of target organ toxicity and to expedite the development of biomarker assays for drug development.
肌球蛋白轻链1（Myl3）是肌球蛋白亚基之一的23 kDa同工型，肌球蛋白是一种参与肌肉收缩的蛋白质。目前，Myl3正作为心脏坏死的生物标志物进行研究，以预测药物诱导的心脏毒性。在本文介绍的工作中，开发并验证了一种液相色谱-串联质谱（LC/MS/MS）测定法，用于测量大鼠血清中的Myl3。该方法的关键步骤包括从血清中免疫亲和纯化Myl3，然后用胰蛋白酶在磁珠上进行消化以释放替代肽。使用合成肽标准品和相应的稳定同位素标记内标对该胰蛋白酶肽进行定量，并将结果按化学计量转换为Myl3血清浓度。对免疫沉淀和消化后的肽回收率（85%）进行校正后得到Myl3浓度，其与合成肽标准品显示出极好的一致性。使用合成肽和His-Myl3蛋白评估测定的准确性（%RE）和精密度（%CV），在3天中的每一天进行测量。合成肽在0.073-7.16 nM范围内进行评估，而在大鼠血清中制备的Myl3蛋白质量控制样品在0.13-6.62 nM范围内进行评估。为制备对照基质，从合并的大鼠血清中免疫去除内源性Myl3。肽的日间准确性和精密度分别不超过7.6%和11.1%，Myl3的日间准确性和精密度分别不超过12.9%和13.2%。本文展示了将该测定法应用于建立时间进程的数据，在该进程中，大鼠在给予异丙肾上腺素后血清Myl3浓度显著升高，异丙肾上腺素是一种已知可诱导心脏损伤的β-肾上腺素能受体激动剂。该测定法是我们实验室更大规模工作的一个实例，即使用LC/MS/MS结合免疫亲和技术来评估靶器官毒性的候选生物标志物，并加快药物开发中生物标志物测定法的开发。
