Toward Comprehensive Analysis of the 3D Chemistry of Pseudomonas aeruginosa Biofilms.
Anna M KotowskaJunting ZhangAlessandro M CarabelliJulie WattsJonathan W AylottIan S GilmorePaul WilliamsDavid J ScurrMorgan R AlexanderPublished in: Analytical chemistry (2023)
Bacterial biofilms are structured communities consisting of cells enmeshed in a self-generated extracellular matrix usually attached to a surface. They contain diverse classes of molecules including polysaccharides, lipids, proteins, nucleic acids, and diverse small organic molecules (primary and secondary metabolites) which are organized to optimize survival and facilitate dispersal to new colonization sites. In situ characterization of the chemical composition and structure of bacterial biofilms is necessary to fully understand their development on surfaces relevant to biofouling in health, industry, and the environment. Biofilm development has been extensively studied using confocal microscopy using targeted fluorescent labels providing important insights into the architecture of biofilms. Recently, cryopreparation has been used to undertake targeted in situ chemical characterization using Orbitrap secondary ion mass spectrometry (OrbiSIMS), providing a label-free method for imaging biofilms in their native state. Although the high mass resolution of OrbiSIMS enables more confident peak assignments, it is still very challenging to assign most of the peaks in the spectra due to complexity of SIMS spectra and lack of automatic peak assignment methods. Here, we analyze the same OrbiSIMS depth profile data generated from the frozen-hydrated biofilm, but employ a new untargeted chemical filtering process utilizing mass spectral databases to assign secondary ions to decipher the large number of fragments present in the SIMS spectra. To move towards comprehensive analysis of different chemistries in the sample, we apply a molecular formula prediction approach which putatively assigns 81% of peaks in the 3D OrbiSIMS depth profile analysis. This enables us to catalog over 1000 lipids and their fragments, 3500 protein fragments, 71 quorum sensing-related molecules (2-alkyl-4-quinolones and N -acylhomoserine lactones), 150 polysaccharide fragments, and glycolipids simultaneously from one data set and map these separated molecular classes spatially through a Pseudomonas aeruginosa biofilm. Assignment of different chemistries in this sample facilitates identification of differences between biofilms grown on biofilm-promoting and biofilm-resistant polymers.
Keyphrases
- candida albicans
- pseudomonas aeruginosa
- biofilm formation
- mass spectrometry
- extracellular matrix
- cystic fibrosis
- label free
- liquid chromatography
- high resolution
- acinetobacter baumannii
- optical coherence tomography
- staphylococcus aureus
- public health
- big data
- healthcare
- density functional theory
- induced apoptosis
- quantum dots
- high resolution mass spectrometry
- water soluble
- escherichia coli
- health information
- ionic liquid
- cell proliferation
- capillary electrophoresis
- ms ms
- preterm infants
- living cells
- cell death
- small molecule
- fatty acid
- social media
- drug discovery
- pi k akt
- tandem mass spectrometry
- data analysis
- endoplasmic reticulum stress
- risk assessment
- oxidative stress
- high density