Roger Karlsson (Presenter)
Sahlgrenska Academy, University of Gothenburg
Bio: I studied chemistry at the University of Gothenburg and chose to focus on analytical chemistry, biochemistry and organic chemistry. My project work was in the field of bioanalytical chemistry and after obtaining my Master degree I continued my Ph.D. studies in the same field. After my Ph.D. studies I have co-founded a biotech company specialized in research on membrane proteins. I have also worked as an Assistant Professor at the Department of chemistry and molecular biology, University of Gothenburg, switching focus to lipidomics and proteomics using liquid chromatography and mass spectrometry. Right now most of my work at the Sahlgrenska University Hospital is about proteotyping, using mass spectrometry and proteomics to find biomarkers for pathogen bacteria directly in clinical samples, without culture.
Authorship: Roger Karlsson (1, 2, 3), Hedvig Engström Jakobsson (2, 3), Francisco Salva Serra (2, 3, 4), Daniel Jaen Luchoro (2, 3, 4), Anders Karlsson (1), Edward R.B. Moore (2, 3, 5)
(1) Nanoxis Consulting AB, Gothenburg, Sweden. (2) Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. (3) Sahlgrenska University Hospital, Clinical microbiology, Gothenburg, Sweden. (4) Universitat de les Illes Balears, Palma, Mallorca, Spain. (5) Culture Collection University of Gothenburg (CCUG), Gothenburg, Sweden.
We explore the use of proteotyping, that is, mass spectrometry and proteomic analysis for rapid and accurate detection of microorganisms, with special interest in pathogenic bacteria. Proteotyping has a higher potential in achieving strain resolution as compared to phenotyping or genotyping, and it can also be used for co-infections. Simultaneously, traits of virulence and antibiotic resistance can be detected. In doing so, the use of broad spectrum antibiotics can be reduced. The ultimate goal is to apply the methodology directly to clinical samples without the need for culturing prior analysis, thus generating a diagnostic protocol, for reliable, rapid, point-of-care diagnostics.
We explore the use of mass spectrometry and proteomic analysis for rapid and accurate detection of microorganisms, with special interest in pathogenic bacteria. Proteotyping, the use of MS-proteomics for characterization and identification of microorganisms, is based on detection of unique peptide biomarkers from proteins (gene expression). The rationale of using tandem mass spectrometry-based proteomics stems from several benefits over traditional methodologies. The proteomic method has a higher potential in achieving strain resolution as compared to phenotyping or genotyping, and it can also be used for mixtures of bacteria (co-infections), whereas MALDI-TOF-MS need pure cultures.
Another benefit of the proposed approach is that markers for the infectious pathogen can be detected simultaneously as detecting traits of virulence and antibiotic resistance. World-wide, increasing development of antibiotic resistant infectious bacteria has been observed, often due to misuse of antimicrobials. Thus, there is an increasingly critical need for new, rapid, accurate and cost-effective techniques for identification of infectious bacteria, for example exhibiting certain virulence traits, as well as the detection of antibiotic-resistant strains. In doing so, the use of broad spectrum antibiotics can be reduced, thus helping to reduce the development of resistance.
Here, we present results from direct proteotyping of clinical samples (nasal swabs, nasopharyngeal swabs).
Sample clean-up. Molysis kit (Molzym, Germany) was used for sample clean-up, removing human biomass (mucus, proteins), while keeping microorganisms intact.
Digestion of proteins into peptides. Bead beating of the bacteria followed by in-solution digestion using trypsin was employed in order to generate peptides.
LC-MS/MS analysis of peptides. Reversed phase nano-LC and QExactive MS (ThermoFisher Scientific) was used for the analysis of the peptides.
Discovery of species specific peptides. In order to identify species specific peptides, an in-house bioinformatics pipeline - TCUP (Typing and Characterization using Proteomics) was used.
Clinical respiratory tract samples (nasal swabs, nasopharyngeal swabs) were analyzed directly (no culture) using the proteotyping approach. First, the clinical samples were screened for common respiratory tract bacterial pathogens such as Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae and Staphylococcus aureus, using an in-house qPCR panel. Proteotyping was then employed on the clinical samples, using both a targeted (peptide biomarkers as inclusion list during analysis) and an open approach (looking for all peptides).
Conclusions & Discussion
Results show that proteotyping is able to detect the infectious pathogen if the numbers of bacterial cells is high enough and that it is possible to see co-infections directly.
The ultimate goal is to apply the methodology directly to clinical samples without the need for culturing the samples prior analysis, thus generating a diagnostic protocol, employing expressed protein biomarker profiling for reliable, rapid, point-of-care diagnostics.
References & Acknowledgements:
IP Royalty: no
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