Federal Institute for Materials Research and Testing (BAM), Division 1.1─Inorganic Trace Analysis, Richard-Willstätter-Str. 11, 12489 Berlin, Germany.
Federal Institute for Materials Research and Testing (BAM), Division 1.7─Organic Trace and Food Analysis, Richard-Willstätter-Str. 11, 12489 Berlin, Germany.
Anal Chem. 2024 Feb 27;96(8):3276-3283. doi: 10.1021/acs.analchem.3c03553. Epub 2024 Jan 31.
We report an analytical methodology for the quantification of sulfur in biological molecules via a species-unspecific postcolumn isotope dilution (online ID) approach using capillary electrophoresis (CE) coupled online with inductively coupled plasma-mass spectrometry (online ID CE/ICP-MS). The method was optimized using a mixture of standard compounds including sulfate, methionine, cysteine, cystine, and albumin, yielding compound recoveries between 98 and 105%. The quantity of sulfur is further converted to the quantity of the compounds owing to the prior knowledge of the sulfur content in the molecules. The limit of detection and limit of quantification of sulfur in the compounds were 1.3-2.6 and 4.1-8.4 mg L, respectively, with a correlation coefficient of 0.99 within the concentration range of sulfur of 5-100 mg L. The capability of the method was extended to quantify albumin in its native matrix (i.e., in serum) using experimentally prepared serum spiked with a pure albumin standard for validation. The relative expanded uncertainty of the method for the quantification of albumin was 6.7% ( = 2). Finally, we tested the applicability of the method on real samples by the analysis of albumin in bovine and human sera. For automated data assessment, a software application (IsoCor)─which was developed by us in a previous work─was developed further for handling of online ID data. The method has several improvements compared to previously published setups: (i) reduced adsorption of proteins onto the capillary wall owing to a special capillary-coating procedure, (ii) baseline separation of the compounds in less than 30 min via CE, (iii) quantification of several sulfur species within one run by means of the online setup, (iv) SI traceability of the quantification results through online ID, and (v) facilitated data processing of the transient signals using the IsoCor application. Our method can be used as an accurate approach for quantification of proteins and other biological molecules via sulfur analysis in complex matrices for various fields, such as environmental, biological, and pharmaceutical studies as well as clinical diagnosis.
我们报告了一种通过毛细管电泳（CE）与电感耦合等离子体质谱（ICP-MS）在线联用的、基于物种非特异性柱后同位素稀释（在线 ID）方法定量生物分子中硫的分析方法。该方法使用包括硫酸盐、蛋氨酸、半胱氨酸、胱氨酸和白蛋白在内的标准化合物混合物进行优化，得到的化合物回收率在 98%至 105%之间。由于事先知道分子中硫的含量，因此将硫的量进一步转换为化合物的量。该方法测定硫的检出限和定量限分别为 1.3-2.6 和 4.1-8.4 mg/L，在 5-100 mg/L 的硫浓度范围内，相关系数为 0.99。该方法的适用性通过用纯白蛋白标准品制备的实验血清对其天然基质（即血清）中的白蛋白进行加标验证来扩展。该方法用于定量白蛋白的相对扩展不确定度为 6.7%（k=2）。最后，我们通过分析牛血清和人血清中的白蛋白来测试该方法在实际样品中的适用性。为了实现自动数据评估，我们在之前的工作中开发了一个软件应用程序（IsoCor），用于处理在线 ID 数据。与以前发表的装置相比，该方法具有以下几个改进：（i）由于特殊的毛细管涂层程序，减少了蛋白质在毛细管壁上的吸附；（ii）通过 CE 在不到 30 分钟内实现了化合物的基线分离；（iii）通过在线装置在一次运行中定量几种硫物种；（iv）通过在线 ID 实现了定量结果的 SI 溯源性；（v）使用 IsoCor 应用程序简化了瞬态信号的数据处理。该方法可用于通过硫分析对复杂基质中的蛋白质和其他生物分子进行定量，适用于环境、生物、制药研究以及临床诊断等多个领域。
