Custom main spectra (MSP) library for the identification of Phytophthora species by MALDI-TOF MS

MALDI-TOF MS is a relatively new technology which has revolutionized the way microorganisms are identified. The method relies on the reproducible detection of microbial protein mass patterns obtained from whole cells, cell lysates, or crude bacterial extracts. Microbial MALDI-TOF mass spectra can be regarded as snapshots of the protein composition of the individual strains studied. The protein biomarkers are typically high-abundance proteins with housekeeping functions, such as ribosomal proteins. For microbial identification mass spectra are analysed by pattern-matching approaches by which mass spectra from the bacteria under study are matching against validated databases of microbial reference spectra. The availability of high-quality and comprehensive spectral databases has been considered of paramount importance for attaining accurate identification results.

Samples:

Culture of 43 selected strains belonging to 26 species of Phytophthora and as an outgroup one species belonging to the genus Pythium were obtained form the Czech Collection of Phytopathogenic Oomycetes (CCPO), https://www.vukoz.cz/index.php/en/collections/collection-of-phytopathogenic-oomycetes-of-rilog

Data collection and processing:

Ethanol/Formic acid extraction sample preparation procedure according to Bruker instructions for use was used. Bruker Bacterial Test Standard (BTS, Bruker, DE) solution was used for calibration. Each spot was measured three times by MALDI-TOF MS Autoflex Speed (Bruker, DE) and flexControl 3.4 (Build 135) with a standard MALDI Biotyper method (MBT_FC.par). Ion source 1 was 19.38 kV, ion source 2 was 18.18 kV, and detection was set from 2 to 20 kDa. For each sample at least 24 spectra were measured, and each spectrum was collected by 2000 shots in 200 steps. For each measurement, the spectra were manually inspected by flexAnalysis 3.4 Compass 1.4 (Bruker Daltonics, DE) and then MSP spectra were processed by standard MALDI Biotyper MSP creation method using MALDI Biotyper Version 3.1 (Build 66).

基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）是一项相对新兴的技术，彻底革新了微生物的鉴定范式。该技术依托于对全细胞、细胞裂解液或粗提细菌提取物中获得的微生物蛋白质质谱模式的可重复检测。微生物的MALDI-TOF质谱可被视为所研究菌株个体蛋白质组成的“快照”。这类蛋白质生物标志物通常为高丰度持家蛋白，例如核糖体蛋白。在微生物鉴定工作中，研究人员通过模式匹配方法分析质谱：将待测细菌的质谱与经过验证的微生物参考光谱数据库进行比对。高质量且覆盖全面的光谱数据库，被认为是获得准确鉴定结果的核心要素。

样本：
从捷克植物病原卵菌保藏中心（Czech Collection of Phytopathogenic Oomycetes, CCPO，https://www.vukoz.cz/index.php/en/collections/collection-of-phytopathogenic-oomycetes-of-rilog）获取了隶属于26个疫霉属物种的43株精选菌株，以及1株作为外类群的腐霉属物种的培养物。

数据采集与处理：
采用依照布鲁克（Bruker）使用说明书制定的乙醇/甲酸萃取样本制备流程。使用布鲁克细菌测试标准品（Bacterial Test Standard, BTS, Bruker, DE）溶液进行质谱校准。每个检测点均通过MALDI-TOF MS Autoflex Speed（Bruker, DE）与flexControl 3.4（Build 135版本），采用标准MALDI Biotyper方法（MBT_FC.par）进行三次重复测量。离子源1电压设置为19.38 kV，离子源2电压设置为18.18 kV，检测质量范围设定为2至20 kDa。每个样本至少采集24张质谱图，每张质谱图通过200个扫描步次、总计2000次激光轰击采集获得。每次测量完成后，均通过flexAnalysis 3.4 Compass 1.4（Bruker Daltonics, DE）对质谱图进行人工检视，随后采用MALDI Biotyper 3.1版本（Build 66）的标准MALDI Biotyper MSP创建方法处理得到主光谱（MSP）。