Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21231, United States.
Departments of Neurology, Johns Hopkins University, Baltimore, Maryland 21205, United States.
J Proteome Res. 2021 Sep 3;20(9):4284-4291. doi: 10.1021/acs.jproteome.1c00238. Epub 2021 Aug 12.
There is a need for targeted analysis of biological fluids for diagnosis, prognosis, or monitoring the progression of diseases. Cerebrospinal fluid (CSF) and serum have been widely used for the development of protein analysis for neurodegenerative diseases and other diseases, respectively. Recently, data-independent acquisition (DIA) mass spectrometry (MS) has been developed to increase the throughput over data-dependent acquisition (DDA) on screening of a large number of samples and discovery of candidate targets. When it comes to target validation, the analytical performance of targeted analysis is critical. However, the inter- and intralaboratory analytical performances of the DIA-MS for targeted proteomic analysis of CSF and serum samples have not yet been investigated. In this study, we showed that the DIA-MS approach allowed us to identify and quantify 1732 CSF and 424 serum proteins, with 90% of proteins identified and quantified in at least 50% of DIA-MS runs. To evaluate the sensitivity, linearity, and dynamic range of the DIA approach, we included the stable isotope-labeled (SI) peptides into CSF and serum samples with serial dilutions. The lower limit of quantification (LLOQ) of peptides was 0.1-0.5 fmol, and the dynamic range was over 3.53 orders of magnitude, with excellent linearity ( < 0.978) in CSF and serum samples. Finally, the reproducibility of the DIA-MS approach was evaluated using entire proteins identified in CSF and serum samples. The intralaboratory three replicate results showed reliable reproducibility with 12.5 and 17.3% of the median coefficient of variation (CV) in both CSF and serum matrices, whereas the median CVs of interlaboratory three replicates were 23.8 and 32.0% in CSF and serum samples, respectively. The comparison of the quantitative result between replicates showed close similarity at intra- and interlaboratories with a median Pearson correlation value of >0.98 in CSF and serum, respectively. In conclusion, we demonstrate the capability of the DIA approach as a targeted proteomic analysis for candidate proteins from CSF and serum samples.
需要针对生物体液进行靶向分析，以用于疾病的诊断、预后或监测。脑脊液 (CSF) 和血清已分别广泛用于开发神经退行性疾病和其他疾病的蛋白质分析。最近，开发了数据非依赖性采集 (DIA) 质谱 (MS) 来增加高通量筛选大量样本和发现候选靶标的能力。在进行目标验证时，靶向分析的分析性能至关重要。然而，CSF 和血清样品的 DIA-MS 靶向蛋白质组学分析的实验室间和实验室内分析性能尚未得到研究。在这项研究中，我们表明 DIA-MS 方法使我们能够鉴定和定量 1732 种 CSF 和 424 种血清蛋白，其中 90%的蛋白在至少 50%的 DIA-MS 运行中被鉴定和定量。为了评估 DIA 方法的灵敏度、线性和动态范围，我们将稳定同位素标记的 (SI) 肽掺入 CSF 和血清样品中，并进行连续稀释。肽的定量下限 (LLOQ) 为 0.1-0.5 fmol，动态范围超过 3.53 个数量级，CSF 和血清样品的线性度极好（<0.978）。最后，使用 CSF 和血清样品中鉴定的整个蛋白质来评估 DIA-MS 方法的重现性。CSF 和血清基质中 12.5%和 17.3%的中位数变异系数 (CV) 的实验室间三重复结果显示出可靠的重现性，而 CSF 和血清样品中实验室间三重复的中位数 CV 分别为 23.8%和 32.0%。在实验室间和实验室内，重复之间的定量结果比较显示出高度相似性，CSF 和血清中的中位数 Pearson 相关值均大于 0.98。总之，我们证明了 DIA 方法作为 CSF 和血清样品候选蛋白的靶向蛋白质组学分析的能力。
