Abstract
Yeasts constitute an oft-neglected class of pathogens among which the resistance to first-line treatments, attributed in part to mutations in efflux pumps, is rapidly emerging. Their thick, chitin-reinforced cell walls render cell lysis difficult, complicating their analysis and identification by methods routinely used for bacteria, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Liquid extraction surface analysis mass spectrometry (LESA-MS) has previously been applied to the analysis of intact proteins from Gram-positive and Gram-negative bacterial colonies sampled directly on solid nutrient media. To date, a similar analysis of yeast colonies has not proved possible. Here we demonstrate the rapid release of intact yeast proteins for LESA-MS by electroporation using a home-built high-voltage device designed to lyse cells grown in colonies on agar media. Detection and identification of previously inaccessible proteins from baker’s yeast Saccharomyces cerevisiae, as well as two clinically relevant yeast species (Candida glabrata and Cryptococcus neoformans), is shown. The electroporation approach also has the potential to be translated to other mass spectrometric analysis techniques, including MALDI and various ambient ionization methods.
Highlights
Yeasts constitute an oft-neglected class of pathogens among which the resistance to first-line treatments, attributed in part to mutations in efflux pumps, is rapidly emerging
It has the capability to lyse bacterial colonies directly on nutrient media and extract their cytosolic proteins,[10−13] an ability which could prove desirable for the identification and characterization of clinical isolates complementary to MALDI-TOF MS, the current gold standard.[14−17] MALDI-TOF MS-based identification of pathogens relies on the comparison of fingerprint protein mass spectra, consisting primarily of ribosomal and housekeeping proteins below 20
Existing protocols designed for MALDI-based workflows recommend extended centrifugation of yeast cultures in 70% formic acid followed by 100% acetonitrile to achieve protein extraction,[20] an approach which was not feasible for liquid extraction surface analysis (LESA) of living colonies
Summary
Yeasts constitute an oft-neglected class of pathogens among which the resistance to first-line treatments, attributed in part to mutations in efflux pumps, is rapidly emerging. Mass spectra generated from yeast colonies following sampling with harsh acetonitrile-based solvent systems were sparse and devoid of peaks attributable to proteins, indicating that lysis was not achieved.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
