Custom MALDI-TOF Mass Spectrometric Database for identification of environmental Burkholderia and related genera

Success of microbial natural products discovery programs is dependent on quick and concise discrimination between isolates from diverse environments. However, laboratory isolation and identification of priority genera using current 16S rRNA PCR-based methods is both challenging and time-consuming. In clinical settings, MALDI-TOF MS is used to rapidly identify unknown bacterial pathogens, yet this has not been widely adapted for isolation of environmental bacteria. To expedite isolation of Gram-negative proteobacterial order Burkholderiales, we have created a main spectral (MSP) library for the Bruker Biotyper MALDI mass spectrometer that includes 95 entries of Burkholderia, Caballeronia, Paraburkholderia, and other environmentally related genera. We tested the effectiveness of the reference library by analyzing 49 unknown environmental isolate strains before comparing to results of traditional 16S sequencing. The MALDI Biotyper correctly identified all strains to the genus level when sufficient reference spectra were available. This dataset is freely available to the scientific research community for use in taxonomic assignments of environmentally-derived bacteria from the order Burkholderiales.

Methods
The Linington MSP Library (Burkholderia).btmsp dataset was acquired on the MALDI Biotyper microflex LT system (Bruker Daltonics). Over 2,250 individual mass spectrometry spectra were acquired in the range of 3,000 to 15,000 m/z to complete the 95 main spectra (MSP) entries. The acquired spectra were processed using associated manufacturer software (see publication for details). Library was exported and packaged in a *.btmsp format for use with MALDI Biotyper systems.

微生物天然产物发掘项目的成功，有赖于对不同环境来源分离菌株的快速精准鉴别。然而，当前基于16S rRNA PCR的实验室分离与重点菌属鉴定方法，不仅操作难度大且耗时良久。在临床场景中，基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）可快速鉴定未知细菌性病原菌，但该技术尚未广泛应用于环境细菌的分离工作。为加快革兰氏阴性变形菌门伯克霍尔德菌目（Burkholderiales）菌株的分离流程，我们为布鲁克（Bruker）Biotyper MALDI质谱仪构建了主光谱（MSP）数据库，该库涵盖伯克霍尔德菌属（Burkholderia）、卡瓦氏菌属（Caballeronia）、副伯克霍尔德菌属（Paraburkholderia）及其他环境相关菌属的95条记录。我们通过分析49株未知环境分离菌株验证了该参考数据库的有效性，并将质谱鉴定结果与传统16S测序结果进行比对。当参考光谱数据充足时，MALDI Biotyper可准确将所有菌株鉴定至属水平。本数据集可向全球科研社区免费开放，用于伯克霍尔德菌目环境来源细菌的分类鉴定工作。

方法
本研究的Linington MSP数据库（伯克霍尔德菌属）.btmsp数据集，采集自MALDI Biotyper microflex LT质谱系统（布鲁克道尔顿，Bruker Daltonics）。实验在3000至15000 m/z的质量范围内采集了超过2250条单张质谱光谱，以完成95条主光谱（MSP）条目。采集得到的光谱通过配套的厂商软件进行处理（详细信息参见已发表文献）。该数据库已导出并封装为*.btmsp格式，可兼容MALDI Biotyper系列质谱系统使用。