Spatially resolved analysis of Pseudomonas aeruginosa biofilm proteomes measured by laser ablation sample transfer

Abstract

Heterogeneity in the distribution of nutrients and oxygen gradients during biofilm growth gives rise to changes in phenotype. There has been long term interest in identifying spatial differences during biofilm development including clues that identify chemical heterogeneity. Laser ablation sample transfer (LAST) allows site-specific sampling combined with label free proteomics to distinguish radially and axially resolved proteomes for Pseudomonas aeruginosa biofilms. Specifically, differential protein abundances on oxic vs. anoxic regions of a biofilm were observed by combining LAST with bottom up proteomics. This study reveals a more active metabolism in the anoxic region of the biofilm with respect to the oxic region for this clinical strain of P. aeruginosa, despite this organism being considered an aerobe by nature. Protein abundance data related to cellular acclimations to chemical gradients include identification of glucose catabolizing proteins, high abundance of proteins from arginine and polyamine metabolism, and proteins that could also support virulence and environmental stress mediation in the anoxic region. Finally, the LAST methodology requires only a few mm2 of biofilm area to identify hundreds of proteins.

Integrating proteomic data with metabolic modelling provides insight into key pathways ofBordetella pertussisbiofilms

“…It would be interesting to see the specific active reactions between the layers of biofilm. Proteomic analysis on layers of biofilm have previously been performed using laser ablation sample transfer (LAST) and label free quantification mass spectrometry (82). A combination of LAST and the methods used in this study may be able to confirm the changes in alanine metabolism.…”

Tissue Sampling and Homogenization in the Sub-Microliter Scale with a Nanosecond Infrared Laser (NIRL) for Mass Spectrometric Proteomics

“…Since in our new approach we ablate directly from the tissue and collect the sample in a reflection configuration, which enables us to sample in three dimensions (3D) we are not only capable of sampling in three dimensions (3D), but also making sure to always sample fully vaporized material. In contrast to a transmission configuration, there is a risk that material above the ablation area will also be blasted off [ 20 ]. Furthermore, we avoid material loss inside the tubing by not using aerosol transport system as described in our previous studies [ 4 , 15 , 16 ].…”

“…Compared to mechanical homogenization, this is a very gentle method of sample extraction and homogenization, avoiding time-consuming preparation steps. Over the past decade, this tissue sampling and homogenization has been successfully demonstrated with a PIRL, nanosecond infrared laser (NIRL) and even a high-energy microsecond infrared laser (MIRL) with subsequent mass spectrometric proteomics [ 4 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ] or directly coupled to real-time MS instruments, such as the “SpiderMass” technology [ 22 , 23 , 24 , 25 , 26 ].…”

Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in Pseudomonas

“…In particular, CF lungs promote the microaerophilic apparatus, including oxidase Cbb3-2, azurin and cytochrome c 551 and the Arc operon encoding for the ADI pathway [83]. More generally, proteomic analysis carried out specifically in the anoxic portion of the biofilm displayed higher abundances of proteins from L-arginine and polyamine metabolism [84]. Chronic P. aeruginosa infections in mammals are regulated by L-arginine and the derived NO metabolism.…”