在线固相萃取-毛细管电泳-质谱法分析癌症血清中的循环microRNA及其转录后修饰,Analytical Chemistry

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在线固相萃取-毛细管电泳-质谱法分析癌症血清中的循环microRNA及其转录后修饰
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-05-07 00:00:00 , DOI: 10.1021/acs.analchem.8b00405
Roger Pero-Gascon ₁ , Victoria Sanz-Nebot ₁ , Maxim V. Berezovski ₂ , Fernando Benavente ₁

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本文介绍了一种在线固相萃取毛细管电泳-质谱（SPE-CE-MS）方法，用于内源性microRNA（miRNA）的纯化，预浓缩，分离和表征以及它们在转录后的修饰。血清。首先，使用hsa-miR-21-5p（miR-21-5p）和hsa-let-7g-5p（let-7g-5p）的标准混合物优化通过CE-MS的分析。对于SPE-CE-MS，使用市售的碳化硅（SiC）树脂制备微型墨盒。在标准条件下优化条件下，微柱寿命（> 25次分析）和重复性（迁移时间的RSD为2.8％； miR-21-5p和let-7g-5p的峰面积分别为4.4％和6.4％RSD）好，方法在25至100nmol·L ^–1之间呈线性关系的检出限（LOD）约为10nmol·L ^–1（比CE-MS低50倍）。为了分析人血清样品，需要在SPE-CE-MS之前进行基于苯酚/氯仿/异戊醇（PCA）提取的离线样品预处理。通过分析来自健康对照和患者的血清样本，证明了SPE-CE-MS方法筛选B细胞慢性淋巴细胞性白血病（CLL）的潜力。仅在CLL患者中检测到microRNA，特别是miR-21-5p和具有3'-尿苷化的23核苷酸长的5'-磷酸化miRNA（iso-miR-16-5p）。

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Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

In this paper, an on-line solid-phase extraction capillary electrophoresis–mass spectrometry (SPE-CE-MS) method is described for the purification, preconcentration, separation, and characterization of endogenous microRNA (miRNA) and their post-transcriptional modifications in serum. First, analysis by CE-MS was optimized using a standard mixture of hsa-miR-21-5p (miR-21-5p) and hsa-let-7g-5p (let-7g-5p). For SPE-CE-MS, a commercial silicon carbide (SiC) resin was used to prepare the microcartridges. Under the optimized conditions with standards, the microcartridge lifetime (>25 analyses) and repeatability (2.8% RSD for the migration times; 4.4 and 6.4% RSD for the miR-21-5p and let-7g-5p peak areas, respectively) were good, the method was linear between 25 and 100 nmol·L^–1, and the limit of detection (LOD) was around 10 nmol·L^–1 (50 times lower than by CE-MS). In order to analyze human serum samples, an off-line sample pretreatment based on phenol/chloroform/isoamyl alcohol (PCA) extraction was necessary prior to SPE-CE-MS. The potential of the SPE-CE-MS method to screen for B-cell chronic lymphocytic leukemia (CLL) was demonstrated by an analysis of serum samples from healthy controls and patients. MicroRNAs, specifically miR-21-5p and a 23 nucleotide long 5′-phosphorylated miRNA with 3′-uridylation (iso-miR-16-5p), were only detected in the CLL patients.

更新日期：2018-05-07

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