Stoppacher Norbert, Reithner Barbara, Omann Markus, Zeilinger Susanne, Krska Rudolf, Schuhmacher Rainer
Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Applied Life Sciences-Vienna, Konrad Lorenz Str. 20, Tulln, Austria.
Rapid Commun Mass Spectrom. 2007;21(24):3963-70. doi: 10.1002/rcm.3301.
Peptaibols are bioactive linear peptides of 5-20 amino acid residues and contain specific non-proteinogenic amino acids such as alpha-aminoisobutyric acid (Aib). They are antibiotic secondary metabolites of moulds belonging predominantly to the genus Trichoderma, some species of which are successfully used as biocontrol organisms to fight against plant diseases. In the present study we developed a profiling method for the relative quantification of 16 trichorzianine peptaibols in culture samples of T. atroviride and the comparison of their expression patterns by liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS). The method is based on selected reaction monitoring (SRM) in a triple-quadrupole tandem mass spectrometer using three SRM transitions per compound. The trichorzianines were enriched by solid-phase extraction (SPE) on C(18) cartridges. SPE recoveries were evaluated for diluted trichorzianine standard solutions and ranged from 72-97%. Suppression of the ionisation of the peptaibols in the ESI source ranged from 67-128% for most of the trichorzianines in culture filtrates of two different strains of T. atroviride and in spiked culture medium. In the case of trichorzianines TA Vb, TA VIa and TA VIb the presence of matrix components in the fungal culture samples caused a reduction of the SRM signal, with intensities between 34% and 56% relative to pure standard solutions. Finally, the profiling method was successfully applied to culture samples of T. atroviride P1 wild-type and two deletion mutants showing different trichorzianine expression patterns characteristic for the investigated fungal strains. This is the first LC-SRM profiling method for peptaibols for the investigation of peptaibol expression patterns in fungal culture samples.
短杆菌肽是由5至20个氨基酸残基组成的具有生物活性的线性肽，包含特定的非蛋白质氨基酸，如α-氨基异丁酸（Aib）。它们是霉菌的抗生素次级代谢产物，主要属于木霉属，其中一些物种已成功用作防治植物病害的生物防治剂。在本研究中，我们开发了一种分析方法，用于相对定量绿色木霉培养样品中的16种木霉短肽，并通过液相色谱/电喷雾电离串联质谱（LC/ESI-MS/MS）比较它们的表达模式。该方法基于三重四极杆串联质谱仪中的选择反应监测（SRM），每个化合物使用三个SRM跃迁。木霉短肽通过在C(18)柱上进行固相萃取（SPE）进行富集。对稀释的木霉短肽标准溶液评估了SPE回收率，范围为72%-97%。在两种不同绿色木霉菌株的培养滤液和加标培养基中，大多数木霉短肽在ESI源中的电离抑制范围为67%-128%。对于木霉短肽TA Vb、TA VIa和TA VIb，真菌培养样品中基质成分的存在导致SRM信号降低，相对于纯标准溶液，强度在34%至56%之间。最后，该分析方法成功应用于绿色木霉P1野生型和两个缺失突变体的培养样品，这两个突变体显示出所研究真菌菌株特有的不同木霉短肽表达模式。这是第一种用于短杆菌肽的LC-SRM分析方法，用于研究真菌培养样品中的短杆菌肽表达模式。
