Podium Presentation in Room 4 on Wednesday at 16:35 (Chair: Kevin Schey / Marissa Jones)
Authors: Emma R. Guiberson (1,2), Daniel J. Ryan (1,2), Andy Weiss (3), Eric P. Skaar (3), Richard M. Caprioli (1,2,4,5,6), Jeffrey M. Spraggins (1,4)
Introduction: Intact protein characterization in situ using MALDI mass spectrometry approaches is challenging owing to limited sequence coverage by traditional tandem mass spectrometry methods of low charge state ions. Additionally, intact protein analysis is generally limited to proteins under 30 kDa. microLESA enables in situ protein identification using liquid extraction surface analysis (LESA) and spatially targeted enzymatic digestion. Here we apply microLESA for the investigation of proteins directly related to the abscess formation in Staphylococcus aureus infection. S. aureus forms abscess communities within infected tissues, but the makeup of these communities is poorly understood. We used microLESA to probe the proteome of the abscess, the leading edge surrounding the abscess, and the cortex, to determine proteomic differences between these three different regions during infection.
Methods: Regions of interest were identified using autofluorescence microscopy based on morphological features. These include the abscess, abscess leading edge, and standard cortex. Microscopy images were then used to guide trypsin deposition by a piezoelectric robotic spotter (Scienion, Berlin, Germany). Trypsin was spotted directly onto regions of interest with a droplet diameter of ~100 µm. Proteolytic peptides were extracted using LESA (Advion, Ithaca, NY), with a droplet diameter between 500 and 1000 µm, and analyzed by LC-MS/MS on an Orbitrap Fusion (Thermo Scientific, Waltham, MA). The microLESA approach was performed on specific regions of S. aureus infected mouse kidney. Samples from 4 and 10 days post-infection were analyzed to highlight time-dependent proteomic differences. Data was searched in Protalizer against known Neumann strain and mouse protein databases.
Results and Discussion: From LC-MS/MS data, 2329 unique proteins were identified (minimum 2 peptides per protein and present in multiple technical and biological replicates), of which 50 were of bacterial origin. Of these, we have identified proteins exclusively observed in specific biological regions including 7 that were associated with the abscess, 11 with the abscess leading edge, and 27 within the normal cortex. By combining spatial and temporal proteomic data, this unique approach was able to elucidate localized pathway information for this infection model. More specifically, when considering only the human proteins observed in the analysis, 21 different pathways were detected. These include known infection and disease pathways as well as the biosynthesis of antibiotics pathway. Selected proteins of interest will be visualized using MALDI imaging mass spectrometry to further elucidate the localization of unique proteins. The microLESA identified proteins, and the ability to probe the proteomic differences across time points in a spatially-resolved manner, provide insight into the dynamic processes of infection and abscess heterogeneity.
|Planning to mention or discuss specific products or technology of the company(ies) listed above:||