"Fix and Click" for Assay of Sphingolipid Signaling in Single Primary Human Intestinal Epithelial Cells.
"Fix and Click" for Assay of Sphingolipid Signaling in Single Primary Human Intestinal Epithelial Cells.
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DOI：
10.1021/acs.analchem.1c03503
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发表时间：
2022-01-25
影响因子：
7.4
通讯作者：
Allbritton NL
中科院分区：
文献类型：
--
作者：
Gallion LA;Wang Y;Massaro A;Yao M;Petersen BV;Zhang Q;Huang W;Carr AJ;Zhang Q;Allbritton NL
毛细管电泳荧光检测法（CE-F）是测定单细胞中酶活性的有效方法。然而，CE-F中使用的细胞酶测定通常利用具有可能影响酶动力学的大体积荧光团的报告底物。为了解决这些挑战，我们描述了一种“固定和点击”的方法，利用炔封端的酶激活报告，基于酶的固定，和点击化学反应，以连接荧光团之前，通过单细胞CE-F分析。利用“固定和点击”策略来研究永生化细胞系和原代人结肠上皮细胞中的鞘脂信号传导。当将鞘氨醇炔报告基因加载到细胞中时，该报告基因代谢为神经酰胺（31.6 ± 3.3%峰面积），而不产生鞘氨醇-1-磷酸。相反，当将报告物鞘氨醇荧光素引入细胞中时，鞘氨醇荧光素转化为鞘氨醇-1-磷酸和下游产物（32.8 ± 5.7%峰面积）而不形成神经酰胺。使用与CE-F配对的“固定和点击”在来自分化的和干/增殖的人结肠上皮的单细胞中测量鞘脂代谢，以突出来自原代人结肠上皮的单细胞中鞘氨醇代谢的多样性。这种新的方法将发现广泛的实用性，单细胞酶测定的性能，凭借其在时间和空间上分开的细胞反应与炔封端的报告，随后在稍后的时间和地点的酶激活的测定的能力。
Capillary electrophoresis with fluorescence detection (CE-F) is a powerful method to measure enzyme activation in single cells. However, cellular enzymatic assays used in CE-F routinely utilize reporter substrates that possess a bulky fluorophore that may impact enzyme kinetics. To address these challenges, we describe a “fix and click” method utilizing an alkyne-terminated enzyme activation reporter, aldehyde-based fixation, and a click chemistry reaction to attach a fluorophore prior to analysis by single-cell CE-F. The “fix and click” strategy was utilized to investigate sphingolipid signaling in both immortalized cell lines and primary human colonic epithelial cells. When the sphingosine alkyne reporter was loaded into cells, this reporter was metabolized to ceramide (31.6 ± 3.3% peak area) without production of sphingosine-1-phosphate. In contrast when the reporter sphingosine fluorescein was introduced into cells, sphingosine fluorescein was converted to sphingosine-1-phosphate and downstream products (32.8 ± 5.7% peak area) without formation of ceramide. Sphingolipid metabolism was measured in single cells from both differentiated and stem/proliferative human colonic epithelium using “fix and click” paired with CE-F to highlight the diversity of sphingosine metabolism in single cells from primary human colonic epithelium. This novel method will find widespread utility for performance of single-cell enzyme assays by virtue of its ability to temporally and spatially separate cellular reactions with alkyne-terminated reporters followed by assay of enzyme activation at a later time and place.
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影响因子：
14.8
作者：
Hinman SS;Wang Y;Kim R;Allbritton NL
通讯作者：
Allbritton NL
影响因子：
6.5
作者：
Lieser, B;Liebisch, G;Schmitz, G
通讯作者：
Schmitz, G
影响因子：
4.6
作者：
Levy, Michal;Futerman, Anthony H.
通讯作者：
Futerman, Anthony H.
DOI：
10.1016/j.jchromb.2021.122739
发表时间：
2021-05-13
影响因子：
3
作者：
Lee, Gwang Bin;Kim, Young Beom;Moon, Myeong Hee
通讯作者：
Moon, Myeong Hee
影响因子：
15
作者：
Himo, F;Lovell, T;Fokin, VV
通讯作者：
Fokin, VV