, Bundesanstalt für Materialforschung und -prüfung (BAM), Richard-Willstätter-Straße 11, 12489, Berlin, Germany.
Charité - Universitätsmedizin Berlin, Hessische Str. 3-4, 10115, Berlin, Germany.
Anal Bioanal Chem. 2022 Jun;414(15):4441-4455. doi: 10.1007/s00216-022-03974-z. Epub 2022 Mar 22.
Quantitative proteomics is a growing research area and one of the most important tools in the life sciences. Well-characterized and quantified protein standards are needed to achieve accurate and reliable results. However, only a limited number of sufficiently characterized protein standards are currently available. To fill this gap, a method for traceable protein quantification using sulfur isotope dilution inductively coupled plasma mass spectrometry (ICP-MS) was developed in this study. Gel filtration and membrane filtration were tested for the separation of non-protein-bound sulfur in the protein solution. Membrane filtration demonstrated a better performance due to the lower workload and the very low sulfur blanks of 11 ng, making it well suited for high-purity proteins such as NIST SRM 927, a bovine serum albumin (BSA). The method development was accomplished with NIST SRM 927e and a commercial avidin. The quantified mass fraction of NIST SRM 927e agreed very well with the certified value and showed similar uncertainties (3.6%) as established methods while requiring less sample preparation and no species-specific standards. Finally, the developed procedure was applied to the tau protein, which is a biomarker for a group of neurodegenerative diseases denoted "tauopathies" including, e.g., Alzheimer's disease and frontotemporal dementia. For the absolute quantification of tau in the brain of transgenic mice overexpressing human tau, a well-defined calibration standard was needed. Therefore, a pure tau solution was quantified, yielding a protein mass fraction of (0.328 ± 0.036) g/kg, which was confirmed by amino acid analysis.
定量蛋白质组学是一个不断发展的研究领域，也是生命科学中最重要的工具之一。为了获得准确可靠的结果，需要具有良好特征和定量的蛋白质标准品。然而，目前只有有限数量的具有足够特征的蛋白质标准品可用。为了填补这一空白，本研究开发了一种使用硫同位素稀释电感耦合等离子体质谱（ICP-MS）进行可追溯蛋白质定量的方法。凝胶过滤和膜过滤均用于分离蛋白质溶液中非蛋白结合的硫。由于工作量低且硫空白仅为 11 ng，膜过滤表现出更好的性能，非常适合高纯度蛋白质，如 NIST SRM 927（牛血清白蛋白（BSA））。该方法的开发是使用 NIST SRM 927e 和一种商业亲和素完成的。NIST SRM 927e 的定量质量分数与认证值非常吻合，并且与已建立的方法具有相似的不确定度（3.6%），同时需要更少的样品制备，并且不需要特定于物种的标准品。最后，该方法应用于 Tau 蛋白，Tau 蛋白是一组神经退行性疾病（称为“tau 病”）的生物标志物，包括阿尔茨海默病和额颞叶痴呆症。为了对过度表达人 Tau 的转基因小鼠脑中的 Tau 进行绝对定量，需要一个定义明确的校准标准品。因此，对纯 Tau 溶液进行了定量，得到的蛋白质质量分数为（0.328±0.036）g/kg，通过氨基酸分析得到了验证。
