Environmental and Plant Biology Department, Athens 45701, USA; Molecular and Cellular Biology Program, Ohio University, Athens 45701, USA.
Chemistry and Biochemistry Department, Athens 45701, USA; Molecular and Cellular Biology Program, Ohio University, Athens 45701, USA.
Plant Sci. 2019 Sep;286:49-56. doi: 10.1016/j.plantsci.2019.04.025. Epub 2019 May 31.
Progress in the functional biochemical analysis of plant glycosyltransferases (GTs) has been slow because plant GTs are generally membrane proteins, operate as part of larger, multimeric complexes, and utilize a vast complexity of substrate acceptors. Therefore, the field would benefit from development of adequate high throughput expression as well as product detection and characterization techniques. Here we review current approaches to tackle such obstacles and suggest a new path forward: nucleic acid programmable protein arrays (NAPPA) with liquid sample desorption ionization (LS-DESI-MS) mass spectrometry. NAPPA utilizes in vitro transcription and translation to produce epitope-tagged fusion proteins from cloned GT cDNAs. LS-DESI is a soft ionization technique that allows rapid and sensitive MS-based product characterization in situ. Coupling both approaches provides the opportunity to examine individual GT functions as well as protein-protein interactions. Furthermore, advances in automated oligosaccharide synthesis and lipid nanodisc technology should allow testing of plant GT activity in presence of numerous substrate acceptors and lipid environments in a high throughput fashion. Thus, NAPPA-DESI-MS has great potential to make headway in biochemical characterization of the large number of plant GTs.
植物糖基转移酶（GTs）的功能生化分析进展缓慢，因为植物 GT 通常是膜蛋白，作为更大的多聚体复合物的一部分发挥作用，并且利用大量复杂的受体底物。因此，该领域将受益于开发足够的高通量表达以及产物检测和鉴定技术。在这里，我们回顾了当前解决这些障碍的方法，并提出了一个新的前进方向：核酸可编程蛋白阵列（NAPPA）与液体样品解吸电离（LS-DESI-MS）质谱。NAPPA 利用体外转录和翻译，从克隆的 GT cDNA 中产生表位标记融合蛋白。LS-DESI 是一种软电离技术，允许在原位快速和灵敏的基于 MS 的产物鉴定。这两种方法的结合提供了研究单个 GT 功能以及蛋白质-蛋白质相互作用的机会。此外，自动化寡糖合成和脂质纳米盘技术的进步应该允许以高通量的方式在存在多种受体底物和脂质环境的情况下测试植物 GT 的活性。因此，NAPPA-DESI-MS 具有在大量植物 GT 的生化特性研究方面取得进展的巨大潜力。
