A new strategy of using O18-labeled iodoacetic acid for mass spectrometry-based protein quantitation.
A new strategy of using O18-labeled iodoacetic acid for mass spectrometry-based protein quantitation.
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DOI：
10.1007/s13361-012-0396-9
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发表时间：
2012-07
影响因子：
3.2
通讯作者：
Kaltashov IA
中科院分区：
文献类型：
--
作者：
Wang S;Kaltashov IA
一个新的O 18标记协议的设计，以协助定量的含半胱氨酸的蛋白质，使用LC/MS。与其他O 18标记策略，标签进行在完整的蛋白质水平（消化前）在半胱氨酸侧链的还原/烷基化过程中使用O 18标记的碘乙酸（IAA）。后者可以很容易地通过交换羧基氧原子的市售IAA在O 18-富集的水在低pH值。由于在蛋白质中的O 18标签的掺入发生在整个蛋白质，而不是肽，水平，定量结果是不依赖于肽。该标签在温和pH条件下的优异稳定性为标记后的样品处理步骤提供了所需的灵活性和耐用性。与通常昂贵的同位素标记试剂相比，这种方法仅使用两种相对便宜的市售试剂（IAA和H2O 18）。使用80 kDa的人血清转铁蛋白（hTf）作为模型，其中线性定量跨越三个数量级的动态范围内实现的新方法的可行性证明。这种新方法可用于定量蛋白质组学应用，特别适用于生物制药领域的各种任务，从药代动力学研究到蛋白质治疗的质量控制。
A new O18 labeling protocol is designed to assist quantitation of cysteine-containing proteins using LC/MS. Unlike other O18 labeling strategies, the labeling is carried out at the intact protein level (prior to its digestion) during reduction/alkylation of cysteine side chains using O18-labeled iodoacetic acid (IAA). The latter can be easily prepared by exchanging carboxylic oxygen atoms of commercially available IAA in O18-enriched water at low pH. Since incorporation of the O18 label in the protein occurs at the whole protein, rather than peptide, level, the quantitation results are not peptide-dependent. The excellent stability of the label in mild pH conditions provides flexibility and robustness needed of sample processing steps following the labeling. In contrast to generally costly isotope labeling reagents, this approach uses only two relatively inexpensive commercially available reagents (IAA and H2O18). The feasibility of the new method is demonstrated using an 80 kDa human serum transferrin (hTf) as a model, where linear quantitation is achieved across a dynamic range spanning three orders of magnitude. The new approach can be used in quantitative proteomics applications and is particularly suitable for a variety of tasks in the biopharmaceutical sector, ranging from pharmacokinetic studies to quality control of protein therapeutics.
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