High sensitivity (zeptomole) detection of BODIPY-labelled heparan sulfate (HS) disaccharides by ion-paired RP-HPLC and LIF detection enables analysis of HS from mosquito midguts.
High sensitivity (zeptomole) detection of BODIPY-labelled heparan sulfate (HS) disaccharides by ion-paired RP-HPLC and LIF detection enables analysis of HS from mosquito midguts.
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DOI：
10.1039/d2ay01803a
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发表时间：
2023-03-16
影响因子：
3.1
通讯作者：
Skidmore, Mark A.
中科院分区：
文献类型：
--
作者：
Maciej-Hulme, Marissa L.;Leprince, Anaelle C. N.;Lavin, Andre;Guimond, Scott E.;Turnbull, Jeremy E.;Pelletier, Julien;Yates, Edwin A.;Powell, Andrew K.;Skidmore, Mark A.
硫酸乙酰肝素（HS）的精细结构，细胞表面和细胞外基质HS蛋白聚糖的糖胺聚糖多糖组分，协调控制稳态和驱动多细胞动物发育的复杂细胞信号传导过程。此外，HS还参与病毒、细菌和寄生虫对哺乳动物的感染。目前荧光标记的HS二糖的检测限（低飞摩尔; 10−15 mol），有效地阻碍了对小的、功能相关的细胞和组织群体中HS组成的研究，这些细胞和组织群体可能阐明感染和其他生化过程的结构要求。在这里，超高灵敏度的方法进行了描述，利用反相HPLC的组合，与四辛基溴化铵（托亚布）作为离子配对试剂和激光诱导荧光检测BODIPY-FL标记的二糖。该方法使检测灵敏度无与伦比地提高了106个数量级，能够在zeptomolar范围内（100 - 21摩尔; <1000个标记分子）进行检测。这有助于从所选组织的微量样品中确定HS二糖组成分析，如通过分析从冈比亚按蚊中肠分离的HS所证明的，其在不接近检测限的情况下实现。离子配对反相高效液相色谱-激光诱导荧光检测法用于肝素和硫酸乙酰肝素的超灵敏二糖成分分析。
The fine structure of heparan sulfate (HS), the glycosaminoglycan polysaccharide component of cell surface and extracellular matrix HS proteoglycans, coordinates the complex cell signalling processes that control homeostasis and drive development in multicellular animals. In addition, HS is involved in the infection of mammals by viruses, bacteria and parasites. The current detection limit for fluorescently labelled HS disaccharides (low femtomole; 10−15 mol), has effectively hampered investigations of HS composition in small, functionally-relevant populations of cells and tissues that may illuminate the structural requirements for infection and other biochemical processes. Here, an ultra-high sensitivity method is described that utilises a combination of reverse-phase HPLC, with tetraoctylammonium bromide (TOAB) as the ion-pairing reagent and laser-induced fluorescence detection of BODIPY-FL-labelled disaccharides. The method provides an unparalleled increase in the sensitivity of detection by ∼six orders of magnitude, enabling detection in the zeptomolar range (∼10−21 moles; <1000 labelled molecules). This facilitates determination of HS disaccharide compositional analysis from minute samples of selected tissues, as demonstrated by analysis of HS isolated from the midguts of Anopheles gambiae mosquitoes that was achieved without approaching the limit of detection. Ultra-sensitive disaccharide compositional analysis of heparin and heparan sulfate facilitated by ion-paired, reversed phase HPLC coupled with laser-induced fluorescence detection.
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DOI：
10.1111/febs.12231
发表时间：
2013-05
期刊：
The FEBS journal
影响因子：
--
作者：
Shao C;Shi X;White M;Huang Y;Hartshorn K;Zaia J
通讯作者：
Zaia J
影响因子：
7.6
作者：
Ghezzi S;Cooper L;Rubio A;Pagani I;Capobianchi MR;Ippolito G;Pelletier J;Meneghetti MCZ;Lima MA;Skidmore MA;Broccoli V;Yates EA;Vicenzi E
通讯作者：
Vicenzi E
影响因子：
4.8
作者：
Frese, Marc-Andre;Milz, Fabian;Dierks, Thomas
通讯作者：
Dierks, Thomas
影响因子：
3.1
作者：
Carruthers, VB;Håkansson, S;Sibley, LD
通讯作者：
Sibley, LD
影响因子：
3.1
作者：
JHA, R;DAVIS, JT
通讯作者：
DAVIS, JT