Mass spectrometry-based 'omic mining through the biostrata of Pseudomonas aeruginosa colonies and biofilms

铜绿假单胞菌是一种革兰氏阴性杆菌，是引起多种医院感染的致病菌。然而，这种微生物最为人所知的可能是在囊性纤维化患者的肺部深处形成对抗生素不敏感的粘液菌落和生物膜，并导致这一人群的高发病率和死亡率。细菌组件，如生物膜，在过去的几十年里一直是深入研究的主题。特别是，它们一直是众多功能基因组分析的焦点。然而，由于生物膜和菌落是复杂的结构，在机械干预下会分解，这一事实阻碍了对这些方法得出的数据的解释。这意味着，由于样本采集过程的侵入性/破坏性，我们必然会丢失大量关于生物分子表达的空间分布的信息。低吞吐量的方法，如激光扫描共聚焦荧光显微镜分析荧光标记的融合蛋白显示，生物膜很可能表现出分层的蛋白质表达。然而，直到最近，还没有一种高通量的方法可以应用于评估全球生物分子谱如何通过和跨越微生物生物组装而变化。直接研究这一问题的可能性最近随着对环境(即“潮湿”)条件下的生物样本的实时、空间分辨分析的引入而来。完成这项工作所需的技术利用了基于解吸电喷雾电离的“DESI”MS。在这里，带电的溶剂液滴的精细喷雾被用来‘开采’到样品中。然后，解吸的离子被收集起来，并进入质谱仪进行在线分析，对于蛋白质来说，还可以进行自上而下的鉴定。Desi-MS是由Graham Cooks博士的实验室开发的，他已经将这项技术应用于一系列生物样本(包括人的皮肤、干的和液体的血液、植物表面、尿样，甚至实体肿瘤的边缘)。Cooks实验室的工作也证明了DESI对于蛋白质组和代谢组学都是有效的。然而，在Cooks的实验室之外，DESI几乎没有被接受，部分原因是必要的硬件最近才可用，部分原因是更广泛的生物科学界--特别是微生物学家--对这项技术缺乏认识。在这个项目中，我们的目标是利用DESI来研究代谢物和蛋白质表达谱如何在铜绿假单胞菌的菌落和生物膜之间变化。我们完全预计，这种方法将提供关于生长在这些结构不同部分的细胞的全球生物分子分布的信息。这种方法非常好地补充了我们现有的研究重点，并将为BBSRC资助的剑桥蛋白质组学中心的能力增加一个Valauble额外维度。

Pseudomonas aeruginosa is a Gram-negative opportuinistic human pathogen, which is responsible for causing a wide range of nosocomial infections. However, the organism is perhaps best known for forming antibiotic-insensitive mucoid colonies and biofilms deep within the lungs of patients with cystic fibrosis, and is responsible for causing high rates of morbidity and mortality among this population. Bacterial assemblies such as biofilms have been the subject of intensive investigation over the last few decades. In particular, they have been the focus of numerous functional genomic analyses. However, interpretation of the data derived from these approaches has been hampered by the fact that biofilms and colonies are complex structures which break down upon mechanical intervention. This means that we necessarily lose a lot of information about the spatial distribution of biomolecule expression simply due to the invasive/destructive nature of the sample harvesting procedure. Low through-put approaches like laser scanning confocal fluorescence microscopic analysis of fluorescently-labelled fusion proteins has revealed that biofilms are highly likely to exhibit stratified protein expression. However, until very recently, no high through-put approach could be applied to assess how global biomolecule profiles vary through and across microbial bioassemblies. The potential to investigate this issue directly came recently with the introduction of real-time, spatially-resolved analysis of biological samples in ambient (i.e., 'wet') conditions. The technology required to do this exploits 'DESI' MS, which is based on desorption electrospray ionization. Here, a fine spray of charged solvent droplets is used to 'mine in' to the sample. The resulting desorbed ions are then collected and passed into a mass spectrometer for on-line analysis, and, in the case of proteins, 'top-down' identification. DESI-MS was developed in the lab of Dr Graham Cooks, who has applied the technique to a range of biological samples (including human skin, dried- and liquid blood, plant surfaces, urine samples and even the margins of solid tumours). Work from Cooks' lab has also demonstrated that DESI is effective for both proteomic and metabolomic profiling. However, outside Cooks' lab, DESI has seen very little uptake, partly because the necessary hardware has only recently become available, and partly due to lack of awarness of the technology amongst the wider biological sciences community - especially microbiologists. In this project, we aim to exploit DESI to study how the metabolite and protein expression profiles vary across and through colonies and biofilms of P. aeruginosa. We fully anticipate that this approach will provide information about the global biomolecule profiles of cells growing in different parts of these structures. This approach complements our existing research priorities extremely well, and will add a valauble extra dimension to the capabilities of the BBSRC-funded Cambridge Centre for Proteomics.