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Collision-Induced Dissociation Mass Spectrometry: A Powerful Tool for Natural Product Structure Elucidation Mass spectrometry is a powerful tool in natural product structure elucidation, but our ability to directly correlate fragmentation spectra to these structures lags far behind similar efforts in peptide sequencing and proteomics. Often, manual data interpretation is required and our knowledge of the expected fragmentation patterns for many scaffolds is limited, further complicating analysis. Here, we summarize advances in natural product structure elucidation based upon the application of collision induced dissociation fragmentation mechanisms. Andrew R. Johnson† and Erin E. Carlson*,†,‡,§ †
Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405, United States Department of Molecular and Cellular Biochemistry, Indiana University, 212 South Hawthorne Drive, Bloomington, Indiana 47405, United States data to be analyzed. Analysis of extracts in a targeted approach, that is, detection of an expected compound, is relatively straightforward as it only requires matching of an observed m/z to a database entry. Untargeted analysis of an extract, however, necessitates a way to parse the tremendous amount of data created. First, known compounds must be identified by database searching, a step known as dereplication, and then the remaining unknowns must be prioritized for further studies. Informatics tools are called upon to make this task tractable and efficient. Examples include comparative tools to highlight atural products (NPs), the secondary metabolites differences among samples,11 similarity among fragmentation produced by plants, bacteria, and fungi, have long been spectra, and thus scaffold,12 and identification of likely exploited for their uniquely privileged structures and degradation or metabolic products.13 1,2 therapeutic benefits (Figure 1). Representing the cumulative Just as genomes and proteomes are now routinely result of nature’s own high-throughput screen,3 NPs are characterized, many hoped that a boon of informatics and thought to occupy a tremendous proportion of chemical analytical tools could similarly simplify metabolomics.14,15 space.4−7 The wide range of functionalities and scaffolds Today, it appears likely that these tools will never match the present in these molecules makes them potentially powerful level of generality achieved in the aforementioned fields, largely therapeutic agents, and it is therefore not surprising that due to the complexity of the materials being analyzed. Unlike approximately two-thirds of current pharmaceutical agents are the linear construction of 4 or 20 monomer units in DNA and either NPs, derivatives, or mimics thereof.8 However, this proteins, respectively, natural products, such as those shown in chemical diversity, along with the large dynamic range of their Figure 1, possess near limitless structural diversity precluding production, makes these fascinating compounds difficult to ready prediction of their structures and the possibility of both purify and characterize. Natural product structure general enrichment and isolation strategies often performed in elucidation often requires the combination of multiple DNA and protein studies. techniques including UV−vis spectroscopy, IR spectroscopy, Despite these challenges, recent work to generate informatics X-ray crystallography, and nuclear magnetic resonance (NMR) tools has been highly successful.20−22 Still, the full elucidation spectroscopy. Historically, mass spectrometry has predomof novel structures frequently relies upon manual data inantly been used for molecular formula determination. interpretation and corresponding NMR-based assessment of However, in recent years there has been a surge in the use of these complex compounds, which are often produced at fragmentation spectra to reveal functionalities, motifs, scaffolds, remarkably low physiological concentrations. Most reports of and even natural product families. novel natural products use mass spectrometry as a confirmatory Mass spectrometry has been increasingly relied upon for its tool to NMR data;23 however, the throughput of mass modularity and relative throughput as compared to other spectrometry warrants increased reliance as a structure spectroscopic techniques such as NMR or IR.9,10 Mass elucidation tool. Additionally, mass spectrometry facilitates spectrometry (MS), coupled to liquid chromatography (LC), characterization of compounds which cannot be purified, either has made possible the characterization of crude extracts within due to degradation24 or relative abundance.25 minutes; detecting hundreds or even thousands of features from a single sample over several orders of magnitude. These Published: July 1, 2015 technical advances have created a new problem: the quantity of ‡
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© 2015 American Chemical Society
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DOI: 10.1021/acs.analchem.5b01543 Anal. Chem. 2015, 87, 10668−10678
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Analytical Chemistry
present as well as the structure of both the precursor and product ions. Myriad studies of fragmentation mechanisms and structure elucidation strategies of natural products based on CID fragmentation have been reported; however, general guidelines for characterizing an unknown natural product structure de novo do not exist. Here, we provide a summary of the steps required to take an unknown molecular feature to a partially or fully assigned natural product structure within the context of a crude or partially purified extract (i.e., purified compound has not been obtained), with focus on CID-based fragmentation studies. While other types of fragmentation exist, CID is possible with the most universally available instrumentation, is most frequently reported in the literature, and is the most common type of fragmentation spectra reported to databases.29,30 Instrumentation and Experimental Design. The instrumentation and basic strategies for the structure elucidation of natural products mirror those required in the fields of proteomics and metabolomics, that is, the analysis of highly complex mixtures of compounds with constituents present over many orders of magnitude. All instrumental components (e.g., chromatographic separation, fragmentation, mass analyzer) have been extensively reviewed elsewhere.31−33 As is the case for the analysis of any cellular extract or secretions, the complexity of the samples often necessitates chromatographic separation prior to ionization, typically utilizing electrospray ionization (ESI) or matrix assisted laser desorption ionization (MALDI),12,34 to minimize ion suppression and adequately resolve low-abundance features. After the ions have been introduced into the instrument, they are analyzed to determine their mass to charge ratio (m/z; Figure 2). These data can be used for molecular formula determination and are often the basis for comparative analysis studies to enable the logging of unknown features. The vast majority of structural information is obtained by further analysis of these precursor ions through MS/MS experiments. The precursor ion is mass-selected and fragmented, and the resulting product ions are analyzed. This is done in either a targeted experiment for examination of predetermined compounds or using untargeted methods, where the n most intense ions (typically 3−15) in the precursor scan are fragmented to globally assess the sample for potentially related molecules. The ion selection window, the m/z range included when an ion is selected for fragmentation, can be extremely important to the quality of the resulting data. A wider window, such as 3 Da, provides good sample coverage with optimal S/N. However, additional experiments with a smaller window and correspondingly lower signal intensity (e.g., 1 Da window) are often required to unambiguously assign the observed fragments to a single precursor ion, instead of a discrete ion observed at a similar m/z value. Ion traps, Q-TOFs, Orbitraps, and Fourier transform ion cyclotron resonance (FTICR) instruments are the most commonly utilized, each with benefits and drawbacks (Table 1). The main compromise between these instruments is one of precision versus speed. The Orbitrap and FTICR are capable of mass accuracies that often enable ready determination of molecular formula (
