Teaching Microbial Identification with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Bioinformatics Tools

Abstract

Ever since the first observation of “animalcules” under a microscope, and the subsequent discovery of microorganisms of myriad size, shape, pigmentation and motility modes, classification in aid of microbial identification is key to understanding inter-relationships between diverse microbes. Combining universal applicability with robustness, 16S rRNA sequencing is the gold standard for microbial typing; however, recent developments in clinical diagnostics have called attention to a shift towards PCR-independent instrumentation and methods given PCR’s requirement for expensive and complex sample preparation. Using ribosomal proteins as biomarkers for evolutionary relatedness, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) - originally developed for the soft ionization of proteins and peptides in proteomics studies - has been successfully applied to identifying bacteria, archaea, fungi and viruses to the species, and, on occasions, sub-species level. Though experimentally proven and increasingly adopted in the clinic, the relatively low-cost (on a per sample basis) and rapid MALDI-TOF MS microbial identification technique, along with its theoretical principles and methodology, is a conspicuous absentee in contemporary microbiology curricula. Motivated by a desire to close the curriculum gap, this article describes a discovery-based activity for teaching microbial identification - using MALDI-TOF MS in combination with open-source genomics and proteomics search tools – while providing tips on mass spectra interpretation and activity implementation for lowering the barrier for classroom adoption. Infused with inquiry-based learning concepts guiding students in identifying microbes from environmental water samples with unknown species diversity, the activity spurs students’ learning by igniting their spirit of inquiry, which leads to better mastery of concepts; a significant departure from conventional laboratory exercises that, in verifying known theory or results, lack the ability to excite students or impart skills for seeking answers to their own questions. Suitable as part of a laboratory sequence complementing bioinformatics, analytical chemistry or life sciences courses, the activity’s modular nature also affords creative content adjustments for catering to differing curricula needs and learning styles. Nor is the exercise restricted to water samples, other samples (with appropriate modifications in inoculation protocols) such as vegetable leaves are also interesting microcosms for performing microbial censuses. Taken together, from sample collection to cultivation and microbial typing, students engage in an integrated exercise designed to impart conceptual knowledge and practical skills – while unlocking the joy of learning and discovery – by answering a research question with unknown answers: what are the microorganisms present in an environmental water sample?