Enrichment and fragmentation approaches for enhanced detection and characterization of endogenous glycosylated neuropeptides.
Enrichment and fragmentation approaches for enhanced detection and characterization of endogenous glycosylated neuropeptides.
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糖基化神经肽是近年来在甲壳类动物中发现的一种神经内分泌系统的模式生物。存在几种工作流程来表征酶消化的肽;然而，内源性神经肽的独特性质需要重新评估方法。我们评估使用亲水相互作用液相色谱（HILIC）富集和不同的片段化方法，以进一步探测在Callinectes sapidus糖基化神经肽的表达。在HILIC的评价过程中，我们观察到内源性肽样品需要含水量较少的溶剂。这种修饰使检测到的神经肽糖型的数量增加了近两倍，从18增加到36。产物离子触发的电子转移/高能碰撞解离使55个完整的N-和O-连接的糖型的位点特异性检测成为可能，而更快的步进碰撞能量高能碰撞解离导致25个的检测。将该工作流程应用于5种神经元组织，能够表征已知神经肽的36种糖型和9种推定的新型神经肽的11种糖型。总体而言，甲壳类动物中的糖基化神经肽的数据库从10个神经肽家族的40种神经肽的18种糖型大大扩展到136种糖型。宏观和微观的异质性进行了观察，证明了这种简单的无脊椎动物的化学多样性，建立了一个框架，使用甲壳类动物来探测糖基化对神经肽的调节作用。
Glycosylated neuropeptides were recently discovered in crustaceans, a model organism with a well-characterized neuroendocrine system. Several workflows exist to characterize enzymatically digested peptides; however, the unique properties of endogenous neuropeptides require methods to be re-evaluated. We evaluate the use of hydrophilic interaction liquid chromatography (HILIC) enrichment and different fragmentation methods to further probe the expression of glycosylated neuropeptides in Callinectes sapidus. During the evaluation of HILIC, we observed the necessity of a less aqueous solvent for endogenous peptide samples. This modification enabled the number of detected neuropeptide glycoforms increased almost two-fold, from 18 to 36. Product ion-triggered electron-transfer/higher-energy collision dissociation enabled the site-specific detection of 55 intact N- and O-linked glycoforms, while the faster stepped collision energy higher-energy collisional dissociation resulted in detection of 25. Applying this workflow to five neuronal tissues enabled the characterization of 36 glycoforms of known neuropeptides and 11 glycoforms of 9 putative novel neuropeptides. Overall, the database of glycosylated neuropeptides in crustaceans was largely expanded from 18 to 136 glycoforms of 40 neuropeptides from 10 neuropeptide families. Both macro- and micro-heterogeneity were observed, demonstrating the chemical diversity of this simple invertebrate, establishing a framework to use crustacean to probe modulatory effects of glycosylation on neuropeptides.
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DOI：
10.1007/s13361-011-0273-y
发表时间：
2012-01-01
影响因子：
3.2
作者：
Hart-Smith, Gene;Raftery, Mark J.
通讯作者：
Raftery, Mark J.
影响因子：
8.6
作者：
Davies, Leela R. L.;Varki, Ajit
通讯作者：
Varki, Ajit
影响因子：
4.8
作者：
Eckmair, Barbara;Jin, Chunsheng;Paschinger, Katharina
通讯作者：
Paschinger, Katharina
DOI：
10.1007/978-1-62703-146-2_10
发表时间：
2013-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Jensen, Pia Honnerup;Mysling, Simon;Jensen, Ole Norregaard
通讯作者：
Jensen, Ole Norregaard
DOI：
10.3791/1207
发表时间：
2009-03-23
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Gutierrez, Gabrielle J;Grashow, Rachel G
通讯作者：
Grashow, Rachel G