Department of Chemistry, Faculty of Natural Sciences and Institute of Chemical Biology (ICB), Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.
MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, UK.
Rapid Commun Mass Spectrom. 2020 Nov 30;34(22):e8904. doi: 10.1002/rcm.8904.
Glycosyl-inositol-phospho-ceramides (GIPCs) or glycosylphosphatidylinositol-anchored fungal polysaccharides are known to be major lipids in plant and fungal plasma membranes and to play an important role in stress adaption. However, their analysis remains challenging due to the several steps involved for their extractions and purifications prior to mass spectrometric analysis. To address this challenge, we developed a rapid and sensitive method to identify GIPCs from the four common fungal plant pathogens Botrytis cinerea, Fusarium graminearium, Neurospora crassa and Ustilago maydis.
Fungal plant pathogens were cultured, harvested, heat-inactivated and washed three times with double-distilled water. Intact fungi were deposited on a matrix-assisted laser desorption ionization (MALDI) target plate, mixed with the matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid solubilized at 10 mg/mL in chloroform-methanol (9:1 v/v) and analyzed using a Bruker MALDI Biotyper Sirius system in the linear negative ion mode. Mass spectra were acquired from m/z 700 to 2000.
MALDI time-of-flight (TOF) mass spectrometric analysis of cultured fungi showed clear signature of GIPCs in B. cinerea, F. graminearium, N. crassa and U. maydis.
We have demonstrated that routine MALDI-TOF in the linear negative ion mode combined with an apolar solvent system to solubilize the matrix is applicable to the detection of filamentous fungal GIPCs.
糖基肌醇磷脂酰基神经酰胺（GIPCs）或糖基磷酸酰基肌醇锚定真菌多糖已知是植物和真菌质膜中的主要脂质，并在应激适应中发挥重要作用。然而，由于在进行质谱分析之前需要进行多个步骤的提取和纯化，因此它们的分析仍然具有挑战性。为了解决这一挑战，我们开发了一种从四种常见的真菌植物病原体（灰葡萄孢菌、禾谷镰刀菌、粗糙脉孢菌和玉米黑粉菌）中鉴定 GIPCs 的快速灵敏方法。
培养真菌植物病原体，收获，热灭活并用水三次洗涤。完整的真菌被沉积在基质辅助激光解吸电离（MALDI）靶板上，与基质混合，基质由 2,5-二羟基苯甲酸和 2-羟基-5-甲氧基苯甲酸以 9:1 的比例混合，在氯仿-甲醇（9:1 v/v）中溶解，浓度为 10mg/mL，并使用 Bruker MALDI Biotyper Sirius 系统在线性负离子模式下进行分析。采集质荷比从 700 到 2000 的质谱。
培养真菌的 MALDI 飞行时间（TOF）质谱分析显示灰葡萄孢菌、禾谷镰刀菌、粗糙脉孢菌和玉米黑粉菌中存在 GIPCs 的明确特征。
我们已经证明，常规 MALDI-TOF 在线性负离子模式下结合非极性溶剂系统来溶解基质可用于检测丝状真菌的 GIPCs。
