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[email protected] Current Metabolomics, 2015, 3, 130-137
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MALDI Mass Spectrometric Imaging of the Nematode Caenorhabditis elegans Robert F. Menger1,#, Chaevien S. Clendinen2,#, Louis A. Searcy1, Arthur S. Edison2 and Richard A. Yost1* 1
Department of Chemistry, University of Florida; 2Department of Biochemistry & Molecular Biology, University of Florida, Gainesville, FL, USA Abstract: Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) is a technique that records mass spectra as a function of position across a biological tissue sample, yielding images of chemical distribution. Until now, MALDI-MSI has typically been performed on thinly sliced tissue sections that are coated with a UV-absorbing matrix. We have developed protocols to apply MALDI-MSI to chemically interrogate intact Caenorhabditis elegans, a nematode approximately 1-mm in length. C. elegans is a model organism with numerous available genetic mutants, three of which were used in this study to validate the MALDI-MSI results using principal component analysis (PCA). In comparison to traditional chemical biology analyses of nematodes that require large-scale cultures, MALDIMSI has the selectivity and sensitivity to record chemically relevant data from analysis of a single worm. This study demonstrates the feasibility of MALDI-MSI as an important new tool to study the chemistry of individual nematodes as well as the potential to conduct chemical biology and metabolomics studies of parasitic species that are impossible to culture outside of the host.
Keywords: MALDI-imaging, MS-imaging, metabolomics, nematode chemical biology, daf-22, fat6;fat7, srf-3.
Caenorhabditis elegans (C. elegans) is a free-living nematode found in soil and compost and is one of the most widely studied organisms in biological and biomedical sciences. Since the initial mapping of genetic mutants that influence behavior by Sydney Brenner over 40 years ago [5], numerous studies have established C. elegans as an important model organism. C. elegans is a self-fertilizing nematode with 959 somatic cells [6]. Its small size (~1 mm in length), short life cycle (3.5 days), and a simple body plan contribute to the ease of laboratory manipulations, which
now have enabled a rich area of research into C. elegans chemical biology [7-9] and metabolomics [10]. These studies typically require large numbers of developmentally synchronized worms grown in liquid culture [9]. Current methodologies for identifying such molecules include the use of pooled samples of nematodes ranging from ~10,000 to 4,000,000, depending on the concentration of the analytes and the analytical technology. In a typical chemical signaling study, exudates (e.g. the exometabolome) are collected and characterized by NMR and LC/MS [11]. A number of mass spectrometry investigations have been conducted using wildtype (N2) C. elegans and readily available gene-knockouts. Many of these investigations have characterized the fatty acid content from fractionated nematode extracts using techniques such as GC-FID or GC-MS [12-16]. A number of significant differences in fatty acid catabolism were reported; however, other small molecules that are inefficiently extracted from the worm may not be observed using such methods. At least two approaches have been demonstrated that utilize chemically selective imaging of C. elegans, a time-of-flight secondary ion mass spectrometry (ToF-SIMS) study that was able to obtain very high resolution data but with relatively low sensitivity [17] and X-ray fluorescence microscopy (XFM) to interrogate the 4th row inorganic metals in fully hydrated C. elegans [18].
*Address correspondence to this author at the Department of Chemistry, University of Florida, Gainesville, FL, USA; Tel: (352)392-0557; Fax: (352) 392-4651; E-mail:
[email protected] # These authors contributed equally to this study
We present an alternative approach using MALDI-MSI for C. elegans chemical biology and metabolomics studies that use small numbers of individual animals rather than pooled samples. There are many advantages of this approach: minimal sample preparation, minimal numbers of
INTRODUCTION Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) is an ideal method to reveal the distribution of biochemicals in tissue [1, 2]. In contrast to other mass spectrometry-based surface analysis techniques such as secondary ion mass spectrometry (SIMS) or laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), MALDI-MS provides a method for soft ionization of biomolecules (e.g., lipids, peptides, and proteins) [3]. Traditional MALDI-MSI methodologies have investigated thin, microtomed tissue sections; however, MALDIMSI has the potential for small organism surface analysis without prior sectioning [4], assuming the organism satisfies the size constraints of typical instruments.
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MALDI Mass Spectrometric Imaging of the Nematode Caenorhabditis elegans
animals needed, and the ability to eventually apply this method to parasitic species that cannot be cultured. To demonstrate the potential for this approach, we have used multivariate analysis to compare MALDI-MSI data from several mutants that were predicted to produce different compositions of small molecule metabolites. METHODS Chemicals and Reagents 2,5-dihydroxybenzoic acid (DHB) was purchased from Acros Organics (Geel, Belgium). 9-aminoacridine (9-AA) was purchased from MP Biomedicals (Solon, OH). HPLCgrade methanol (MeOH), HPLC-grade water (H2O), and sodium acetate (NaOAc) were purchased from Fisher Scientific (San Jose, CA). 100% ethanol (EtOH) was purchased from Decon Labs (King of Prussia, PA). DHB was dissolved in 70:30 MeOH:H2O (v/v) to a final concentration of 40 mg/mL with a final concentration of 10 mM NaOAc added to serve as a MALDI matrix. Similarly, 9-AA was dissolved in 70:30 EtOH:H2O (v/v) to a final concentration of 6 mg/mL; however, NaOAc was not added to the 9-AA matrix solution. MALDI-MS of Nematodes Synchronized young adult C. elegans, N2 and mutants, were washed three times in distilled H2O and then pipetted onto a Fisherfinest Premium microscope glass slide and allowed to dry. C. elegans were coated with the prepared MALDI matrix (either DHB or 9-AA) by pneumatic spraying with a Type A glass Meinhard nebulizer (Golden, CO). Matrix solution was delivered using a flow rate of 3.0 mL/min and nitrogen was used as a nebulization gas at 30 psi. Care was taken to avoid excess wetting during matrix deposition. Three passes were conducted over the microscope slide at a height of approximately 10 cm before allowing a drying time of approximately 15 seconds. The process was repeated until 8 mL of MALDI matrix solution was deposited atop the microscope slide. All MALDI-MS experiments were conducted in positive ion mode utilizing a Thermo Scientific LTQ XL linear ion trap mass spectrometer (San Jose, CA) equipped with a Thermo MALDI ionization source. The MALDI ionization source consisted of a Lasertechnik Berlin MNL 106-LD N2 laser (Berlin, Germany). The 337 nm laser was operated at a repetition rate of 60 Hz and produced a laser spot size of approximately 100 μm in diameter. The source region of the instrument was maintained at a pressure of 75 mTorr. A laser energy of 4.5 μJ and 3 laser shots per laser stop were utilized for an individual scan. Automatic gain control (AGC) was toggled off during analysis in order to maintain a fixed number of laser shots at each position along the nematode.
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resulting from the MALDI matrix (DHB or 9-AA). Mass-tocharge (m/z) and intensity lists were exported from the Thermo Qualbrowser and read into MATLAB (R2012a) for processing and analysis. Once imported, major matrix peaks were removed, and PCA and PLS-DA were performed to determine major sample outliers, which were also removed. The data were then normalized via probabilistic quotient and log scaled for PCA and PLS-DA. Following normalization and removal of major outliers, 2-dimensional scores plots were generated to visualize the separation of sample groupings. Loadings, which are the m/z values that underlie the relationships between the mass spectra given by the scores, were then analyzed to determine important features. A t-test with Bonferonni correction was also conducted to verify significance of these features. MALDI-MSI and Tandem MS Following multivariate data analysis, MS imaging was conducted over select nematodes. Compound identification for ions with high loading values was conducted using tandem MS with collision-induced dissociation (CID). In these experiments, MS2 imaging experiments were conducted over the entire nematode. MS2 spectra were collected on the nematodes using a raster step size of 25 μm (oversampling). In all tandem MS experiments, a laser energy of 4.5 μJ and 3 laser shots per spot were utilized. Additionally, the collision energy was maintained at 35 AU (arbitrary units normalized to m/z 400). Following data collection, MS2 images were generated for intense fragment ions. Only those ions that were found to localize on the nematodes were used for analyte identification. RESULTS In this study, we have utilized MALDI-MS to characterize the metabolic profiles of individual whole C. elegans. To validate the approach, we have compared four different genotypes: laboratory reference (N2) and mutant strains daf22, fat6;fat7, and srf-3. The daf-22 mutant lacks a gene that encodes a sterol carrier protein (SCPx) homologue, which catalyzes the final step in peroxisomal fatty acid -oxidation, cleaving carbons from long-chain fatty acid side chains [19]. Thus, daf-22 mutants may accumulate long-chain fatty acid lipids. The fat-6;fat-7 mutants lack two 9 desaturase isoforms and thus demonstrate a decreased overall fatty acid content due to increased fatty acid catabolism [20, 21]. The fat-6 and fat-7 genes encode stearoyl-CoA desaturases and thus desaturate stearic acid (18:0) [20]. Mutations in fat-6 and fat-7 result in a complete lack of 20-carbon polyunsaturated fatty acids as well as oleic acid (18:19) and its derivatives [21]. Srf-3, included in the supplementary data, encodes a nucleotide sugar transporter and mutants demonstrate a reduction in O- and N-linked glycoconjugates containing UDP-galactose [22, 23].
Data Processing and Analysis MALDI-MS spectra were collected over the length of each nematode. In doing so, ten mass spectra were averaged from head to tail to generate one sample MS spectrum with each nematode analyzed in triplicate. Furthermore, mass spectra from areas off the nematodes were collected in triplicate during each experiment to determine background peaks
MALDI-MS Spectra are Different for Different Genotypes MALDI-MS was utilized to characterize the compounds naturally occurring in both N2 and mutant genotypes. Care was taken to obtain average mass spectra along the length of the nematodes in order to eliminate spectral variation that
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may occur due to the area sampled. On average, each nematode was approximately 1 mm in length; thus, 10 mass spectra were required to sample the entire length of the nematode assuming a laser spot diameter of 100 μm. Representative MS spectra from the N2 and the mutant nematodes (daf-22 and fat6;fat7) are shown in (Fig. 1). Analysis of the nematodes coated with DHB demonstrated a number of MALDI matrix-related ions (e.g., m/z 154, 231, 273, and 313), all of which are labeled with an asterisk in (Fig. 1A and B). Although matrix-related ions are generally uninformative, the N2 animals exhibited a relatively higher abundance of m/z 231, putatively identified as a
Menger et al.
potassium adduct with DHB. Since a potassium salt was not added to the matrix solution, this matrix ion may reflect the potassium content of the nematode. Additionally, a number of phosphatidylcholine (PC) lipid ions were observed in both the N2 and daf-22 spectra, namely m/z 184, 542, and 580, identified as the [M+H]+ of the PC headgroup, the [M+H]+ of lysophosphatidylcholine (LPC) 20:5, and the [M+K]+ of LPC 20:5. In general, the daf-22 demonstrated a relatively higher abundance for both the protonated ion and the potassium adduct of LPC 20:5. As compared to DHB, the use of 9-AA (Fig. 1C and D) as a MALDI matrix produced strikingly different mass spec-
Fig. (1). Representative MS spectra utilizing DHB as a MALDI matrix from (A) the N2 and (B) the daf-22 mutant cuticles, and representative MS spectra utilizing 9-AA as a MALDI matrix from (C) the N2 and (D) and the fat6;fat7 mutant nematodes. Ions known to result from the MALDI matrix are labeled with an asterisk. Each spectrum is an average of 10 MS scans.
MALDI Mass Spectrometric Imaging of the Nematode Caenorhabditis elegans
tra from the nematodes. In particular, 9-AA demonstrated a significantly lower matrix background in positive mode, with only one appreciable matrix ion at m/z 195. The basicity of 9-AA (pKa~10) is thought to be responsible for this low background; however, the basicity also limits the number of compounds that can be efficiently ionized in positive mode; analytes with a preformed positive charge (e.g., choline and carnitine containing analytes) are among the exceptions. A comparison of the mass spectra from the N2 and fat6;fat7 mutants is shown in (Fig. 1C and D). The N2 spectrum produced a number of abundant lyso and intact PCs, with the most abundant ion at m/z 808, corresponding to the [M+Na] + of PC (18:1/18:1). In contrast, the fat6;fat7 mutants demonstrated low signal from both the lyso and intact PCs, and demonstrated relatively high ion signal from m/z 258, 280, and 296, identified as the [M+H]+, [M+Na] +, and [M+K]+, respectively, of glycerophosphocholine (GPC). Principal Component Analysis Clearly Differentiates Worm Genotypes PCA revealed clear separation between the N2 and mutant nematodes (Fig. 2A and C). The quality of this separation depends significantly upon the type of microscope slide and MALDI matrix used. The reason for this phenomenon is not yet clear, but using a Fisherfinest Premium microscope glass slide yielded the best separation in all scenarios. In regards to the MALDI matrix, DHB with 10 mM sodium acetate yielded the best separation for N2 and daf-22 mu-
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tants; however, the best separation for N2 and fat6;fat7 mutants resulted from the use of 9-AA. The loadings plots shown in (Fig. 2B and D) reveal the mass spectral variation between N2 and mutants. The direction of the peaks on the loadings plot indicates the ions loaded with each strain. The negative, blue peaks correlate with the N2, whereas the positive, red peaks correlate with the specified mutant. Each feature is colored according to the loading coefficients.19 According to PCA, the MS of the daf-22 nematodes has a higher abundance of high mass compounds compared to the N2. As stated previously, daf-22 accumulates long-chain fatty acids, and this correlates with the difference in mass distribution in the loadings plot. The fat6;fat7 mutants are deficient in C20 mono- and polyunsaturated fatty acids [21], and thus masses associated with such lipids, most notably, PC (18:1/20:5), correlate strongly with the N2s (p
