Simple Silica Column-Based Method to Quantify Inorganic Polyphosphates in Cartilage and Other Tissues
Simple Silica Column-Based Method to Quantify Inorganic Polyphosphates in Cartilage and Other Tissues
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DOI：
10.1177/1947603517690856
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发表时间：
2018-10-01
期刊：
影响因子：
2.8
通讯作者：
Kandel, Rita A.
中科院分区：
文献类型：
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作者：
Lee, Whitaik David;Gawri, Rahul;Kandel, Rita A.
Objective.无机多磷酸盐（polyP）在哺乳动物生物学中发挥着多种作用。PolyP的研究受到缺乏简单而灵敏的定量方法的阻碍。本研究的目的是开发一种稳健的方法，用于定量哺乳动物组织（如软骨）中低水平的polyP，该组织富含干扰其测定的大分子。设计用蛋白酶K消化天然和体外形成的组织以释放隔离的聚P。将组织消化物加载到二氧化硅旋转柱上，然后洗脱结合的聚P，并评估各种处理以使非聚P荧光最小化。然后使用基于聚P发生的DAPI（4 '，6-二脒基-2-苯基吲哚）荧光位移的荧光测定法定量洗脱液的聚P含量。结果蛋白酶K预处理降低了蛋白质对polyP恢复的抑制作用。洗脱液被核酸和糖胺聚糖污染，导致外来荧光信号。然后通过核酸酶处理和添加浓缩的Tris缓冲液有效地消除这些。使用掺有已知量的聚P的样品定量聚P水平并确定回收率。该二氧化硅旋转柱方法能够回收至少80%的初始负载的聚P，并且检测低至10（-10）mol.结论.这种灵敏、重复性好、易于操作的聚P定量方法将成为研究哺乳动物细胞和组织中聚P生物学的有用工具。虽然该方案是针对哺乳动物组织开发的，但该方法应该能够定量大多数生物来源中的polyP，包括血液和血清等液体样品。
Objective. Inorganic polyphosphates (polyP) play a multitude of roles in mammalian biology. PolyP research is hindered by the lack of a simple and sensitive quantification method. The aim of this study was to develop a robust method for quantifying the low levels of polyP in mammalian tissue such as cartilage, which is rich in macromolecules that interfere with its determination. Design. Native and in vitro formed tissues were digested with proteinase K to release sequestrated polyP. The tissue digest was loaded on to silica spin columns, followed by elution of bound polyP and various treatments were assessed to minimize non-polyP fluorescence. The eluent was then quantified for polyP content using fluorometry based on DAPI (4',6-diamidino-2-phenylindole) fluorescence shift occurring with polyP. Results. Proteinase K pretreatment reduced the inhibitory effect of proteins on polyP recovery. The eluent was contaminated with nucleic acids and glycosaminoglycans, which cause extraneous fluorescence signals. These were then effectively eliminated by nucleases treatment and addition of concentrated Tris buffer. PolyP levels were quantified and recovery ratio determined using samples spiked with a known amount of polyP. This silica spin column method was able to recover at least 80% of initially loaded polyP, and detect as little as 10(-10) mol. Conclusions. This sensitive, reproducible, easy to do method of quantifying polyP will be a useful tool for investigation of polyP biology in mammalian cells and tissues. Although the protocol was developed for mammalian tissues, this method should be able to quantify polyP in most biological sources, including fluid samples such as blood and serum.