Incorporating LC–MS/MS Analysis and the Dereplication of Natural Product Samples into an Upper-Division Undergraduate Laboratory Course

Abstract

Growth in the biomedical and biotechnology sectors requires a highly trained and highly skilled workforce to answer the next great scientific questions. Undergraduate laboratory courses incorporating hands-on training based in authentic research position soon-to-be graduates to learn in environments that mirror those of academic, industrial, and government laboratories. Mass spectrometry is one of the most broadly applied analyses carried out in the biomedical and pharmaceutical sciences, and thus, it is essential that upper-division students gain hands-on experience in techniques and analytical workflows in mass spectrometry. Our precourse assessments identified weaknesses in student experience and knowledge in the fundamentals of mass spectrometry, supporting that it was a necessary area for improvement. We incorporated a laboratory experiment focused on tandem mass spectrometry and database searching into a preexisting mini-semester project devoted to identifying metabolites from medicinal plants. Implementation of the experiment allowed students to make more confident metabolite identifications, introduced them to a cutting-edge database analysis platform (GNPS: Global Natural Products Social Molecular Networking), and increased student experience and knowledge of mass spectrometry in addition to the principle of dereplication of samples derived from nature.

Mass Spectrometry Does Not Need to Be a “Grind”: Plant Secondary Metabolites in Coffee and Black Pepper as a Practical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Teaching Tool

“…If a standard of a compound of interest is available, quantitative analysis and comparisons could also easily be incorporated into this module (see instructor notes, SI File 4). The number and diversity of the alkamides identified also suggests that a laboratory module like this even could be adapted for "crowdsourced" MS/MS-based discovery of novel secondary metabolites (perhaps emphasizing a dereplication approach as in Jouaneh et al 18 ).…”

“…Very recently, Jouaneh and co-workers published a laboratory experiment where LC-MS/MS and database searching were used to "dereplicate" (e.g., identify known compounds) in medicinal plant extracts. 18 Despite these recent reports, the analysis of secondary metabolites in native plant extracts and consumer products appears relatively uncommon as a teaching tool, despite MS/MS finding powerful utility in secondary metabolite discovery and structural elucidation. To this end, we developed a two-part upper-division undergraduate laboratory module dedicated to the LC-MS/MS analysis of two classes of secondary metabolites in plant-derived foods: chlorogenic acids in coffee and alkamides in peppercorns.…”

“…In a similar report, Manninen et al developed a laboratory module employing selected-reaction monitoring (SRM, which is another term for MRM) to identify the different red anthocyanin pigments extracted from autumn leaves, which differed only by the presence of different hydroxyl and methoxyl groups. Very recently, Jouaneh and co-workers published a laboratory experiment where LC-MS/MS and database searching were used to “dereplicate” (e.g., identify known compounds) in medicinal plant extracts …”

Dereplication of tropane alkaloids from four Erythroxylum species using liquid chromatography coupled with ESI‐MSn and HRESIMS

“…8,9 One of the indispensable techniques in the process of dereplication of known natural products is mass spectrometry, specifically liquid chromatography coupled to tandem mass spectrometry, which has greater reliability through the analysis and comparison of the fragmentation patterns of metabolites. [9][10][11] In this context, some studies have reported the fragmentation of tropane alkaloids, which hinders the dereplication of this important class of metabolites. [12][13][14] Due to the importance of tropane alkaloids for Erythroxylum species, and the fact that many of these metabolites have already been reported in the scientific literature, the use of guided…”

“…Using these techniques, metabolites are identified in a sample by comparing the spectral information of its chemical structure with that of natural products already described in scientific literature and databases, avoiding the isolation process for undesirable compounds 8,9 . One of the indispensable techniques in the process of dereplication of known natural products is mass spectrometry, specifically liquid chromatography coupled to tandem mass spectrometry, which has greater reliability through the analysis and comparison of the fragmentation patterns of metabolites 9–11 . In this context, some studies have reported the fragmentation of tropane alkaloids, which hinders the dereplication of this important class of metabolites 12–14 …”