Kildgaard Sara, Mansson Maria, Dosen Ina, Klitgaard Andreas, Frisvad Jens C, Larsen Thomas O, Nielsen Kristian F
Department of Systems Biology, Technical University of Denmark, Soeltofts Plads 221, Kgs. Lyngby DK-2800, Denmark.
Mar Drugs. 2014 Jun 20;12(6):3681-705. doi: 10.3390/md12063681.
In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]+ ions.
在药物发现过程中，对已知化合物进行可靠且快速的去重复对于鉴定新型生物活性化合物至关重要。在此，我们展示了一种综合方法，该方法使用超高效液相色谱 - 二极管阵列检测 - 四极杆飞行时间质谱仪（UHPLC - DAD - QTOFMS），可同时提供精确质量全扫描质谱（MS）和串联高分辨率质谱（MS/HRMS）数据。该方法在来自曲霉属、青霉属和拟青霉属生物活性海洋来源菌株的化合物上得到了验证，这些化合物包括小聚酮化合物、非核糖体肽、萜类化合物和杂萜类化合物。然后将MS/HRMS数据与一个包含约1300种化合物的内部MS/HRMS库进行比对，以进行明确鉴定。全扫描MS数据用于对不在MS/HRMS库中的化合物进行去重复，并结合紫外/可见光（UV/Vis）和MS/HRMS数据以更快地排除数据库搜索结果。这导致鉴定出了已知抗癌化合物曲霉芬amate的四种新型异构体。除了非常低强度的峰外，使用MS/HRMS方法未发现假阴性结果，该方法被证明对于由系统过载或锁质量丢失导致的数据质量不佳具有稳健性。仅对于像展青霉素这样的小聚酮化合物，明确鉴定需要保留时间和UV/Vis光谱。对于蛇孢菌素家族，其许多结构相似的类似物部分共洗脱，通过结合MS/HRMS数据和[M + Na]+离子的m/z可以正确地归属峰。
