Absolute Quantitation of Low Abundance Plasma APL1β peptides at Sub-fmol/mL Level by SRM/MRM without Immunoaffinity Enrichment
Absolute Quantitation of Low Abundance Plasma APL1β peptides at Sub-fmol/mL Level by SRM/MRM without Immunoaffinity Enrichment
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DOI：
10.1021/pr4010103
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发表时间：
2014-02-01
影响因子：
4.4
通讯作者：
Tomonagat, Takeshi
中科院分区：
文献类型：
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作者：
Sano, Shozo;Tagami, Shinji;Tomonagat, Takeshi
选择/多反应监测（SRM/MRM）已广泛用于特定蛋白质/肽的定量，尽管对复杂样品（例如血浆/血清）中的低丰度蛋白质/肽进行定量仍然具有挑战性。为了克服这个问题，需要富集靶蛋白/肽，例如免疫沉淀;然而，这是劳动密集型的，并且抗体的产生非常昂贵。在这项研究中，我们试图定量血浆低丰度APLP 1衍生的A β样肽（APL 1 β），阿尔茨海默病的替代标志物，SRM/MRM使用稳定的同位素标记的参考肽没有免疫亲和富集。Cibacron Blue染料介导的白蛋白去除和乙腈提取的组合，然后进行C-18强阳离子交换多阶段Tip纯化，用于去除血浆蛋白和不必要的肽。在三重四重质谱仪上开发了最佳和验证的前体离子到APL 1 β的碎片离子跃迁，并优化了用于肽分离的纳米液体色谱梯度，以最大限度地减少血浆的生物干扰。使用稳定的同位素标记（SI）肽作为内部对照，血浆APL 1 β肽的绝对浓度可以定量为几百阿莫尔/mL。据我们所知，这是SRM/MRM定量的内源性血浆肽的最低检测水平。
Selected/multiple reaction monitoring (SRM/MRM) has been widely used for the quantification of specific proteins/peptides, although it is still challenging to quantitate low abundant proteins/peptides in complex samples such as plasma/serum. To overcome this problem, enrichment of target proteins/peptides is needed, such as immunoprecipitation; however, this is labor-intense and generation of antibodies is highly expensive. In this study, we attempted to quantify plasma low abundant APLP1-derived A beta-like peptides (APL1 beta), a surrogate marker for Alzheimer's disease, by SRM/MRM using stable isotope-labeled reference peptides without immunoaffinity enrichment. A combination of Cibacron Blue dye mediated albumin removal and acetonitrile extraction followed by C-18-strong cation exchange multi-StageTip purification was used to deplete plasma proteins and unnecessary peptides. Optimal and validated precursor ions to fragment ion transitions of APL1 beta were developed on a triple quadruple mass spectrometer, and the nanoliquid chromatography gradient for peptide separation was optimized to minimize the biological interference of plasma. Using the stable isotope-labeled (SI) peptide as an internal control, absolute concentrations of plasma APL1 beta peptide could be quantified as several hundred amol/mL. To our knowledge, this is the lowest detection level of endogenous plasma peptide quantified by SRM/MRM.