Comparison of Internal Standard Approaches for SRM Analysis of Alpha-Synuclein in Cerebrospinal Fluid,Journal of Proteome Research

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Comparison of Internal Standard Approaches for SRM Analysis of Alpha-Synuclein in Cerebrospinal Fluid
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2017-12-21 00:00:00 , DOI: 10.1021/acs.jproteome.7b00660
Patrick Oeckl ₁ , Petra Steinacker ₁ , Markus Otto ₁

Affiliation

Absolute protein quantification by selected reaction monitoring (SRM, also MRM) is an alternative to immunoassays, and the gold standard here is the addition of stable-isotope labeled (SIL) proteins (PSAQ). Cerebrospinal fluid (CSF) is the preferred source of biomarkers for neurological diseases, and recent improvements in mass spectrometry enable the quantification of disease-relevant proteins in CSF. We used alpha-synuclein SRM to investigate alternatives to the PSAQ approach in human CSF regarding precision and accuracy, including SIL peptides, winged SIL (WiSIL) peptides, and quantitative protein epitope signature tags (QPrESTs). All approaches yielded precise results in CSF with CV values <15% in several runs for all four measured peptides. PSAQ and QPrEST also showed good accuracy (deviation ≤15%), whereas SIL and WiSIL peptides yielded deviations up to 54% that greatly depended on the measured peptide. Total protein concentration in CSF did not affect precision and accuracy. Thus, our study indicates that all four approaches are suitable for relative quantification of alpha-synuclein in CSF. QPrESTs are a valuable alternative to PSAQ in terms of precision and accuracy, although SIL and WiSIL peptides can yield accurate results as well when peptides are selected consciously.

中文翻译：

脑脊液中α-突触核蛋白SRM分析内标方法的比较

通过选择性反应监测（SRM，也称为MRM）对蛋白质进行绝对定量是免疫测定的替代方法，此处的金标准是添加稳定同位素标记的（SIL）蛋白质（PSAQ）。脑脊液（CSF）是神经系统疾病的生物标记物的首选来源，最近在质谱技术方面的改进使CSF中疾病相关蛋白的定量成为可能。我们使用α-突触核蛋白SRM来研究人CSF中PSAQ方法的准确性和准确性的替代方法，包括SIL肽，有翼SIL（WiSIL）肽和定量蛋白表位特征标签（QPrESTs）。对于所有四个测得的肽，所有方法在几次运行中均获得了CSF精确结果，CV值<15％。PSAQ和QPrEST也显示出良好的准确性（偏差≤15％），而SIL和WiSIL肽产生的偏差高达54％，这在很大程度上取决于测得的肽。脑脊液中总蛋白浓度不影响精密度和准确性。因此，我们的研究表明，所有四种方法均适用于CSF中α-突触核蛋白的相对定量。就准确性和准确性而言，QPrEST是PSAQ的有价值的替代品，尽管有意识地选择肽时SIL和WiSIL肽也可以产生准确的结果。

更新日期：2017-12-21

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