Coupling of a vented column with splitless nanoRPLC-ESI-MS for the improved separation and detection of brain natriuretic peptide-32 and its proteolytic peptides.
Coupling of a vented column with splitless nanoRPLC-ESI-MS for the improved separation and detection of brain natriuretic peptide-32 and its proteolytic peptides.
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DOI：
10.1016/j.jchromb.2009.02.040
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发表时间：
2009-04-01
影响因子：
3
通讯作者：
Muddiman, David C.
中科院分区：
文献类型：
--
作者：
Andrews, Genna L.;Shuford, Christopher M.;Burnett, John C., Jr.;Hawkridge, Adam M.;Muddiman, David C.
充血性心力衰竭的生物标志物--脑(B型)利钠肽(BNP-32)的循环浓度是用基于酶联免疫吸附试验的检测方法测定的，以便快速诊断和监测疾病进展。最近，随着新的研究表明循环中可能存在多种具有免疫反应能力的BNP，这些检测方法所提供的缺乏分子特异性的问题受到了质疑。为了更好地了解BNP-32与充血性心力衰竭相关的分子生物学，因此使用傅立叶变换离子回旋共振质谱仪等检测平台将是有利的。这种高分辨率质谱仪可以提供无与伦比的分子特异性，并可以方便地识别和表征所有疾病状态下的各种分子形式。不幸的是，BNP在低浓度下循环(低至3fmoles/ml)。因此，要克服该仪器平台的实际检测极限与BNP-32及其替代分子形式的生理水平之间的脱节，将需要许多正交前端技术的共同努力。在这里，我们开始优化这些前端技术，首先提高在线纳米LC-ESI-MS分离BNP-32及其蛋白降解片段的条件。通过对各种层析参数的广泛分析，我们确定Michrom Magic C8固定相与连续的通风柱配置结合使用可为涉及完整的BNP-32及其相关的胰蛋白酶多肽的纳米流分离提供先进的层析性能。此外，还对胰酶消化BNP-32的条件进行了研究。我们证明，使用游离半胱氨酸作为烷基化猝灭剂，并在消化方案中进行二次消化，可以提供丰度更高的靶向胰蛋白酶多肽。这些数据结合在一起，将有助于进一步加强LC-MS对BNP-32的检测。
The circulating concentration of a biomarker for congestive heart failure, Brain (B-type) Natriuretic Peptide (BNP-32), is measured using ELISA based assays in order to rapidly diagnose and monitor disease progression. The lack of molecular specificity afforded by these assays has recently come into question as emerging studies indicate there are potentially multiple heterogeneous forms of BNP in circulation with immunoreactive capabilities. In order to better understand the molecular biology of BNP-32 as it relates to congestive heart failure, it would thus be advantageous to use a detection platform such as Fourier transform ion cyclotron resonance mass spectrometry. This high resolving power mass spectrometer can provide unparalleled molecular specificity and can facilitate identification and characterization of the various molecular forms across all disease states. Unfortunately, BNP circulates at low concentrations (as low as 3 fmoles/mL). Thus, it will require a collaborative effort from a number of orthogonal front-end technologies to overcome the disconnect between the practical detection limits of this instrument platform and the physiological levels of BNP-32 and its alternative molecular forms. Herein, we begin optimization of these front-end techniques by first enhancing the conditions for online nanoLC-ESI-MS separations of BNP-32 and its proteolytic fragments. Through extensive analysis of various chromatographic parameters we determined that Michrom Magic C8 stationary phase used in conjunction with a continuous, vented column configuration provided advanced chromatographic performance for the nano-flow separations involving intact BNP-32 and its associated tryptic peptides. Furthermore, conditions for the tryptic digestion of BNP-32 were also studied. We demonstrate that the use of free cysteine as an alkylation quenching agent and a secondary digestion within the digestion scheme can provide targeted tryptic peptides with increased abundances. Combined, these data will serve to further augment the detection of BNP-32 by LC-MS.
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影响因子：
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作者：
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影响因子：
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DOI：
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DOI：
10.1073/pnas.0508782102
发表时间：
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影响因子：
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