Comparison of a stable isotope labeled (SIL) peptide and an extended SIL peptide as internal standards to track digestion variability of an unstable signature peptide during quantification of a cancer biomarker, human osteopontin, from plasma using capillary microflow LC-MS/MS
Comparison of a stable isotope labeled (SIL) peptide and an extended SIL peptide as internal standards to track digestion variability of an unstable signature peptide during quantification of a cancer biomarker, human osteopontin, from plasma using capillary microflow LC-MS/MS
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DOI：
10.1016/j.jchromb.2015.05.040
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发表时间：
2015-09-15
影响因子：
3
通讯作者：
Karnes, H. Thomas
中科院分区：
文献类型：
--
作者：
Faria, Morse;Halquist, Matthew S.;Karnes, H. Thomas
人骨桥蛋白（hOPN）是一种分泌的血浆蛋白，其在各种癌症中升高并且指示不良预后。在这里，我们描述了涉及扩展肽内标物的调查，以跟踪不稳定的签名肽，然后进一步的方法开发和验证的定量测量hOPN从血浆中使用微流液相色谱和串联质谱法（MFLC-MS/MS）。使用生物学相关胰蛋白酶肽“GDSVVYGLR”作为该方法的特征肽。优化的方法包括使用hOPN特异性抗体免疫捕获分析物蛋白，然后胰蛋白酶消化以获得特征肽。在沃茨IonKey/MS系统上使用2.5 μ L/min的流速进行分析。使用免疫捕获缓冲液作为验证研究的替代基质。在25-600 ng/mL的k范围内验证了该方法。批内和批间准确度在+/-13%范围内。批内和批间精密度均在17%以内。评价稳定同位素标记（SIL）肽GDSVVYGLR* 和扩展SIL肽TYDGRGDSVV*YGLRSKSKKE作为内标物（IS），以说明特征肽消化的不稳定性和变异性。固有的消化变异性，即，在受控条件下，与两种IS肽相比在+/-20%以内。在消化变异性研究中，胰蛋白酶活性变化（20-180%），使用扩展SIL肽作为内标物将变异性限制在标准化响应的+/-30%范围内。或者，当SIL肽用作内标物时，变异性范围为归一化响应的-67.4%至50.6%。通过从10名健康个体和10名乳腺癌患者获得的血浆样品中定量OPN来证明经验证的方法的适用性。健康个体的血浆OPN浓度范围为38至85 ng/mL，平均浓度为55.4 +/- 15.3 ng/mL。在乳腺癌患者样品中观察到OPN浓度增加1.5-12倍，范围为85至637 ng/mL。(C)2015爱思唯尔B. V.保留所有权利。
Human osteopontin (hOPN) is a secreted plasma protein which is elevated in various cancers and is indicative of poor prognosis. Here we describe investigations involving an extended peptide internal standard to track an unstable signature peptide followed by further method development and validation for quantitative measurement of hOPN from plasma using microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide 'GDSVVYGLR' was used as a signature peptide for this method. The optimized method involved immunocapture of the analyte protein using hOPN specific antibodies followed by trypsin digestion to obtain the signature peptide. Analysis was carried out on a Waters IonKey/MS system using a flow rate of 2.5 mu L/min. Immunocapture buffer was used as a surrogate matrix for the validation studies. The method was validated over a krange of 25-600 ng/mL. Intra-assay and inter-assay accuracies were within +/-13%. Intra-assay and interassay precision were within 17%. A stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKE were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. Inherent digestion variability i.e., under controlled conditions, was within +/-20% with both IS peptides. In digestion variability studies, where trypsin activity was varied (20-180%), the use of the extended SIL peptide as an internal standard limited the variability to within +/-30% of the normalized response. Alternatively, when the SIL peptide was used as the internal standard, the variability ranged from -67.4% to 50.6% of the normalized response. The applicability of the validated method was demonstrated by quantification of OPN from plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. The plasma OPN concentrations in healthy individuals ranged from 38 to 85 ng/mL with a mean concentration of 55.4 +/- 15.3 ng/mL. A 1.5-12 fold increase in OPN concentrations, ranging from 85 to 637 ng/mL, was seen in breast cancer patient samples. (C) 2015 Elsevier B.V. All rights reserved.