Grauso Laura, Mariggiò Stefania, Corda Daniela, Fontana Angelo, Cutignano Adele
Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy.
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, via Pietro Castellino 111, 80131 Napoli, Italy.
PLoS One. 2015 Apr 10;10(4):e0123198. doi: 10.1371/journal.pone.0123198. eCollection 2015.
The glycerophosphoinositols constitute a class of biologically active lipid-derived mediators whose intracellular levels are modulated during physiological and pathological cell processes. Comprehensive assessment of the role of these compounds expands beyond the cellular biology of lipids and includes rapid and unambiguous measurement in cells and tissues. Here we describe a sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the most abundant among these phosphoinositide derivatives in mammalian cells, the glycerophosphoinositol (GroPIns). The method has been developed in mouse Raw 264.7 macrophages with limits of quantitation at 3 ng/ml. Validation on the same cell line showed excellent response in terms of linear dynamic range (from 3 to 3,000 ng/ml), intra-day and inter-day precision (coefficient of variation ≤ 7.10%) and accuracy (between 98.1 and 109.0%) in the range 10-320 ng/ml. As proof of concept, a simplified analytical platform based on this method and external calibration was also tested on four stimulated and unstimulated cell lines, including Raw 264.7 macrophages, Jurkat T-cells, A375MM melanoma cells and rat basophilic leukemia RBL-2H3 cells. The results indicate a wide variation in GroPIns levels among different cell lines and stimulation conditions, although the measurements were always in line with the literature. No significant matrix effects were observed thus indicating that the here proposed method can be of general use for similar determinations in cells of different origin.
甘油磷酸肌醇构成一类具有生物活性的脂质衍生介质，其细胞内水平在生理和病理细胞过程中受到调节。对这些化合物作用的全面评估超出了脂质细胞生物学的范畴，还包括在细胞和组织中进行快速且明确的测量。在此，我们描述了一种灵敏且简便的液相色谱 - 串联质谱法（LC-MS/MS），用于定量分析哺乳动物细胞中这些磷酸肌醇衍生物中最丰富的甘油磷酸肌醇（GroPIns）。该方法是在小鼠Raw 264.7巨噬细胞中开发的，定量限为3 ng/ml。在同一细胞系上的验证表明，在10 - 320 ng/ml范围内，线性动态范围（从3到3000 ng/ml）、日内和日间精密度（变异系数≤7.10%）以及准确度（在98.1%至109.0%之间）方面均表现出色。作为概念验证，基于该方法和外标法的简化分析平台还在四种刺激和未刺激的细胞系上进行了测试，包括Raw 264.7巨噬细胞、Jurkat T细胞、A375MM黑色素瘤细胞和大鼠嗜碱性白血病RBL - 2H3细胞。结果表明，不同细胞系和刺激条件下GroPIns水平存在广泛差异，尽管测量结果始终与文献一致。未观察到明显的基质效应，因此表明本文提出的方法可普遍用于不同来源细胞的类似测定。
