Strategic use of immunoprecipitation and LC/MS/MS for trace-level protein quantification: Myosin light chain 1, a biomarker of cardiac necrosis
Strategic use of immunoprecipitation and LC/MS/MS for trace-level protein quantification: Myosin light chain 1, a biomarker of cardiac necrosis
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DOI：
10.1021/ac070051f
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发表时间：
2007-06-01
影响因子：
7.4
通讯作者：
Ackermann, Bradley L.
中科院分区：
文献类型：
--
作者：
Berna, Michael J.;Zhen, Yuejun;Ackermann, Bradley L.
肌球蛋白轻链1（Myl 3）是肌球蛋白亚基之一的23-kDa同种型，肌球蛋白是参与肌肉收缩的蛋白质。目前正在研究Myl 3作为心脏坏死的生物标志物以预测药物诱导的心脏毒性，并且在本文提出的工作中，开发并验证了LC/MS/MS测定以测量大鼠血清中的Myl 3。该方法中的关键步骤涉及从血清中免疫亲和纯化Myl 3，然后用胰蛋白酶进行珠上消化以释放替代肽。使用合成肽标准品和相应的稳定同位素标记的内标物对该胰蛋白酶肽进行定量，并将结果化学计量转换为Myl 3血清浓度。在免疫沉淀和消化后，针对肽回收率校正Myl 3浓度（85%），并显示出与合成肽标准品的极好一致性。合成肽和His-Myl 3蛋白均用于评价测定准确度（% RE）和精密度（% CV），在3天中的每一天测量。在0.073-7.16 nM的范围内评价合成肽，而在大鼠血清中制备的Myl 3蛋白质QC样品在0.13-6.62 nM的范围内评价。为了制备对照基质，从合并的大鼠血清中免疫耗竭内源性Myl 3。肽的日间准确度和精密度分别不超过7.6%和11.1%，Myl 3日间准确度和精密度分别不超过12.9%和13.2%。提供了来自应用该测定以建立时间过程的数据，在该时间过程中，大鼠在施用异丙肾上腺素（已知诱导心脏损伤的β-肾上腺素能受体激动剂）后显示Myl 3血清浓度显著增加。该测定是我们实验室更大努力的一个例子，使用LC/MS/MS结合免疫亲和技术来评价靶器官毒性的候选生物标志物，并加快药物开发的生物标志物测定的开发。
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.