Relative Mass Defect Filtering of Mass Spectra: A Path to Discovery of Plant Specialized Metabolites

The rapid identification of novel plant metabolites and assignments of newly discovered substances to natural product classes present the main bottlenecks to defining plant specialized phenotypes. Although mass spectrometry provides powerful support for metabolite discovery by measuring molecular masses, ambiguities in elemental formulas often fail to reveal the biosynthetic origins of specialized metabolites detected using liquid chromatography-mass spectrometry. A promising approach for mining liquid chromatography-mass spectrometry metabolite profiling data for specific metabolite classes is achieved by calculating relative mass defects (RMDs) from molecular and fragment ions. This strategy enabled the rapid recognition of an extensive range of terpenoid metabolites in complex plant tissue extracts and is independent of retention time, abundance, and elemental formula. Using RMD filtering and tandem mass spectrometry data analysis, 24 novel elemental formulas corresponding to glycosylated sesquiterpenoid metabolites were identified in extracts of the wild tomato Solanum habrochaites LA1777 trichomes. Extensive isomerism was revealed by ultra-high-performance liquid chromatography, leading to evidence of more than 200 distinct sesquiterpenoid metabolites. RMD filtering led to the recognition of the presence of glycosides of two unusual sesquiterpenoid cores that bear limited similarity to known sesquiterpenes in the genus Solanum. In addition, RMD filtering is readily applied to existing metabolomics databases and correctly classified the annotated terpenoid metabolites in the public metabolome database for Catharanthus roseus.Plant metabolic networks generate amazing chemical diversity, but our understanding of the genetic factors responsible for plant chemistry remains primitive. The discovery and identification of metabolites has posed the greatest bottleneck in recent efforts to exploit metabolomics to address questions about the basis for biosynthetic diversity in the plant kingdom (Ji et al., 2009; Zhou et al., 2012). Since the specialized metabolism of nonmodel plants is taxonomically restricted, metabolite databases offer a poor representation of plant chemical diversity, and de novo recognition and discovery of metabolite chemistry is necessary. A common strategy for metabolite discovery has often started with the generation of tandem mass spectrometry (MS/MS) spectra, usually beginning with the most abundant metabolites, and uses characteristic fragment ions to assign metabolites to a particular class of compounds. Flavonoid identification from MS/MS spectra is often successful because most flavonoids yield MS/MS fragment ions characteristic of their flavonoid cores (Ma et al., 1997; Li et al., 2013). However, when MS/MS spectra fail to display class-characteristic fragment ions, the recognition of a metabolite’s structural class is less obvious.Specialized plant metabolites are often grouped as polyphenolic, terpenoid, alkaloid, polyketide, or fatty acid metabolites based upon the biosynthesis of their core scaffolds, which often undergo subsequent metabolic decoration such as glycosylation. Among phytochemicals, terpenoids offer perhaps the greatest structural diversity. This feature makes them useful as chemical defenses and as the foundation for candidate drugs (Ajikumar et al., 2008; Goodger and Woodrow, 2011), and the commercial importance of terpenes makes their discovery and synthesis an important research focus (Zwenger and Basu, 2008). Terpenoids exhibit remarkable structural diversity resulting from varied metabolic cyclizations, oxidations, rearrangements, and branching reactions (Chappell, 1995; Mizutani and Ohta, 2010) and from diversity in glycosylation (Dembitsky, 2006; Goodger and Woodrow, 2011). Such structural diversity challenges investigators to recognize novel terpenoids in a complex matrix (Pfander and Stoll, 1991; Fraga, 2012), because few features in the MS/MS spectra of nonvolatile terpenoids provide reliable keys for their annotation as terpenoids. As a result, nonvolatile terpenoids represent an underappreciated group of plant specialized metabolites.Advances in chromatography and mass spectrometry (MS) have enabled the detection of a broad range of natural products, and characteristic ions in mass spectra have been useful for distinguishing compound classes. While gas chromatography-MS has enabled the identification of volatile and semivolatile terpenes for decades, it is not a suitable approach for nonvolatile conjugated terpenoids unless they are first cleaved to form volatile products or derivatized to increase volatility. Furthermore, MS/MS fragment ions characteristic of terpenoid glycosides have yet to be documented, and the characterization of conjugated terpenoids has been limited largely to saponins that share a common steroidal or triterpenoid core (Challinor and De Voss, 2013). In contrast with other specialized metabolite classes, the diversity of terpenoid cores dictates that fragment ions specific to terpenoids often fail to provide for the universal recognition of metabolites within this class, particularly for two situations: (1) when terpenoids are glycosylated and MS/MS spectra are dominated by fragment ions derived from the carbohydrate, and (2) when mass spectra are generated in negative ion mode, which often yields limited cleavage of carbon-carbon bonds in the terpenoid core that might serve as terpenoid indicators. The structural diversity of the terpenoid cores yields different fragments in MS/MS spectra of different nonvolatile terpenoids, as has been demonstrated for a series of saponins (Huhman and Sumner, 2002). Therefore, annotations of terpene glycosides in a metabolite profile have been driven by the absence of fragment ions in mass spectra that represent other classes of molecules (Ward et al., 2011).Despite its limited capabilities in differentiating stereoisomers, MS plays important roles in the discovery of natural products and the elucidation of their structures (Lei et al., 2011). Modern medium- to high-resolution mass spectrometers have provided greater (low-ppm) mass measurement accuracy. Such mass measurement errors may be more pronounced than measurements for an individual sample when they represent an average mass extracted from large metabolomics data sets. For metabolites of relatively low molecular mass, such measurements provide sufficient information to assign molecular formulas, but for metabolites of higher (greater than 500 D) molecular masses, formula assignments often are ambiguous owing to the large number of formulas consistent with a molecular mass (Kind and Fiehn, 2007). Moreover, assignments of molecular formulas often fail to yield reliable assignments of metabolites to specific biosynthetic origins.In this report, we examine specialized metabolites of the wild tomato Solanum habrochaites LA1777, which has been studied extensively for its plant defense compounds, including volatile sesquiterpenoids and acyl sugars (Coates et al., 1988; Ghosh et al., 2014). Our recent discovery of a few glycosylated sesquiterpenoids in this accession suggested the metabolic capacity to form such metabolites in the genus (Ekanayaka et al., 2014). It is the intent of this report to present a framework for the accelerated discovery of terpenoid glycosides from mass spectra generated using common instruments such as time-of-flight mass spectrometers that provide intermediate mass resolution and low-ppm mass accuracy using S. habrochaites LA1777 as an example.