Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow.
Absolute quantification of prion protein (90-231) using stable isotope-labeled chymotryptic peptide standards in a LC-MRM AQUA workflow.
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DOI：
10.1007/s13361-012-0411-1
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发表时间：
2012-09
影响因子：
3.2
通讯作者：
Li L
中科院分区：
文献类型：
--
作者：
Sturm R;Sheynkman G;Booth C;Smith LM;Pedersen JA;Li L
大量证据表明，疾病相关的朊病毒蛋白（PrPTSE）的构象构成的病原体在朊病毒疾病。这些疾病影响多种哺乳动物物种。PrPTSE能够将正常朊病毒蛋白（PrPC）的构象转化为对蛋白酶K消化具有抗性的富含β折叠的形式。常见的免疫学技术缺乏在亚飞摩尔水平检测PrPTSE的灵敏度，而动物生物测定、细胞培养和体外转化测定提供超灵敏度，但缺乏免疫学测定提供的高通量。质谱法是上述测定的有吸引力的替代方法，因为它提供了蛋白质的特征肽的高通量、直接测量，通常具有亚飞摩尔的灵敏度。虽然已经报道了用于PrPTSE的液相色谱-多反应监测（LC-MRM）方法，但特征肽的化学组成和缺乏氨基酸序列保守性可能会影响其准确性，并使其难以应用于多个物种。在这里，我们证明了一种替代蛋白酶（胰凝乳蛋白酶）可以产生适用于LC-MRM绝对定量（AQUA）实验的签名肽。新方法提供了几个优点，包括：（1）胰凝乳蛋白酶特征肽缺乏可能混淆测定准确度的化学活性残基（Cys，Met）;（2）低阿托摩尔检测限和定量限（LOD和LOQ）;和（3）在大多数天然易受朊病毒感染的哺乳动物以及重要的实验室模型中保留相同氨基酸序列的特征肽。据作者所知，这是在LC-MRM AQUA工作流程中使用非胰蛋白酶肽的首次报告。
Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiological agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at sub-femtomole levels while animal bioassays, cell culture, and in vitro conversion assays offer ultrasensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein’s signature peptide, often with sub-femtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors’ knowledge, this is the first report of the use of a non-tryptic peptide in a LC-MRM AQUA workflow.
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影响因子：
6.7
作者：
Agrimi U;Nonno R;Dell'Omo G;Di Bari MA;Conte M;Chiappini B;Esposito E;Di Guardo G;Windl O;Vaccari G;Lipp HP
通讯作者：
Lipp HP
影响因子：
11.8
作者：
Joly, DO;Ribic, CA;Samuel, MD
通讯作者：
Samuel, MD
影响因子：
82.9
作者：
Castilla, J;Saá, P;Soto, C
通讯作者：
Soto, C
影响因子：
3.8
作者：
Georgsson, Gudmundur;Sigurdarson, Sigurdur;Brown, Paul
通讯作者：
Brown, Paul
影响因子：
6.7
作者：
Johnson, Christopher J.;Phillips, Kristen E.;Pedersen, Joel A.
通讯作者：
Pedersen, Joel A.