Acid–base-driven matrix-assisted mass spectrometry for targeted metabolomics Rohit Shroffa, Lubomı´rRulı´sˇekb,c, Jan Doubsky´ a, and Alesˇ Svatosˇa,c,1 aMass Spectrometry Research Group, Max Planck Institute for Chemical Ecology, Hans-Kno¨ll-Strasse 8, D-07745 Jena, Germany; and bGilead Sciences and Institute of Organic Chemistry and Biochemistry Research Center, cInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo na´meˇ stı´2, 166 10, Prague 6, Czech Republic Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved April 29, 2009 (received for review January 27, 2009) The ability to charge huge biomolecules without breaking them of our matrices in performing targeted metabolomic studies is apart has made matrix-assisted laser desorption/ionization demonstrated. (MALDI) mass spectrometry an indispensable tool for biomolecular analysis. Conventional, empirically selected matrices produce Results and Discussion abundant matrix ion clusters in the low-mass region (<500 Da), Brønsted–Lowry Concept Was Successfully Applied for the Selection hampering the application of MALDI-MS to metabolomics. An of Ionless Matrices with the Potential to Study a Wide Variety ionization mode of MAILD, a rational protocol for matrix selection of Metabolites. Conventional matrices such as 4-hydoxy-␣- ␣ based on Brønsted–Lowry acid–base theory and its application to cyanocinnamic acid ( -CHCA) and 2,5-dihydroxybenzoic acid Ͻ metabolomics, biological screening/profiling/imaging, and clinical (DHB) produce copious interfering ions in the m/z 500 Th range, diagnostics is illustrated. Numerous metabolites, covering impor- making detection of metabolites in that range rather difficult, if not tant metabolic pathways (Krebs’ cycle, fatty acid and glucosinolate impossible (Fig. 1 A, B, D, and E). We reasoned that mixing an acidic or basic analyte with a strong base or acid, respectively, used biosynthesis), were detected in extracts, biofluids, and/or in bio- as a matrix and crystallization of the salt formed on a target, logical tissues (Arabidopsis thaliana, Drosophila melanogaster, followed by desorption of the formed salt by a laser pulse having Acyrthosiphon pisum, and human blood). This approach moves frequency similar to the absorption maximum of base/acid matrix, matrix selection from ‘‘black art’’ to rational design and sets a should produce ions that are oppositely charged to those of the paradigm for small-molecule analysis via MALDI-MS. absorbing partner [Fig. 1 C and F and supporting information (SI) Fig. S1]. To explore this concept, we investigated ion pairs of 4 clinical diagnostic ͉ matrix design ͉ positive/negative ions ͉ aromatic bases with aliphatic acids monitoring the formation of ion-formation model ͉ MALDI anions using TOF mass spectrometry. The first experiments were performed with a ‘‘proton sponge’’ (15, 16), which is a strong base wing to their ‘‘soft’’ nature of ionization, MALDI-MS (1, 2) [pKa of its conjugate acids 12.5 (15)], 1,8-bis(dimethylamino)naph- Oand electrospray ionization- mass spectrometry (3) (ESI-MS) thalene (DMAN), never previously used as a matrix. Analytes (fatty have been at the forefront of bioanalytical research with far- acids, fatty acid–amino acid conjugates and other anionic species) reaching applications in proteomics (4), genomics (5), biological were mixed in a 1:1 molar ratio with DMAN in ethanol and allowed imaging (6), and metabolomics (7). In MALDI-MS, biomolecules to dry on a MALDI metallic target. Under both vacuum and mixed with matrices (small, UV-absorbing compounds) and ex- atmospheric pressure, gas-phase anions were formed upon UV- laser irradiation (337 or 355 nm) and detected at physiologically posed to laser pulses form gas-phase ions that are typically mea- relevant concentrations (Fig. S2). Negligible fragmentation was sured in time-of-flight (TOF) mass analyzers. Although the high- observed except in polyhydroxylated compounds such as water throughput nature of MALDI-MS makes it an ideal tool for losses in prostaglandin E1 (Fig. S2G). In contrast to common large-scale metabolomic studies, its application in the field has been MALDI matrices, no matrix ions were detected. The absence of rather limited. This is because all conventional matrices (8–10) matrix ions in mass spectra affords a crucial advantage over existing Ͻ produce a forest of interfering low-mass ions ( 500 Da) obscuring matrices. To ensure that we were observing a matrix-assisted the detection of metabolites in the range. Despite several ap- process and not laser desorption/ionization (LDI), mixtures of an proaches (11–13), challenging MALDI-MS-based metabolomic aliphatic base (triethylamine) and an inorganic base (KOH) with tasks such as direct biofluid analysis, on-tissue metabolite screen- strong aliphatic acids (e.g., trifluoroacetic acid) were made and ing, and generation of snapshots of the metabolic machinery of tested for the presence of anion signals. No analyte signal was biological systems remains an unmet challenge. observed even at ablative laser fluences, thus confirming the These limitations call for matrices devoid of interfering ions importance of the UV-absorbing partner in the ionization process. (‘‘ionless matrices’’), yet still assisting an efficient ionization/ desorption of the analytes. An ideal solution would be to have a Potential of Ionless Matrices for Targeted Metabolomics Was Dem- rational selection protocol for such matrices, whereby depending on onstrated on Plant, Insect, and Blood Samples. In situ profiling of a the properties of the analytes of interest, appropriate matrices could damaged Arabidopsis thaliana leaf spotted with DMAN solution be designed. Such a development would not only cross a long-lasting hurdle of empirical selection of MALDI matrices but would also Author contributions: R.S., J.D., and A.S. designed research; R.S. and L.R. performed provide a powerful, fast, and easy-to-use tool to the biological research; A.S. contributed new reagents/analytic tools; R.S., L.R., J.D., and A.S. analyzed community to selectively probe into the metabolomes of living data; and R.S. and A.S. wrote the paper. organisms. Conflict of interest statement: The principal part of the article was filled as US patent Here, we report on a first-ever rational selection protocol for application. matrix-assisted mass spectrometry matrices based on the classical This article is a PNAS Direct Submission. Brønsted–Lowry acid–base theory (14) and density functional Freely available online through the PNAS open access option. theory (DFT) quantum chemical calculations. The matrices devel- Data deposition: The atomic coordinates have been deposited in the Cambridge Structural oped herein are ionless, in other words, the matrices produce no Database (accession no. FO3376). interfering matrix- related ions, thus overcoming the problem of 1To whom correspondence should be addressed. E-mail: [email protected]. most conventional matrices and allowing the detection of small This article contains supporting information online at www.pnas.org/cgi/content/full/ molecules (0–1,000 Da). Furthermore, the enormous applicability 0900914106/DCSupplemental. 10092–10096 ͉ PNAS ͉ June 23, 2009 ͉ vol. 106 ͉ no. 25 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900914106 Downloaded by guest on September 27, 2021 pooled extracts of Drosophila melanogaster males and virgin females showed distinct sex-related profiles (Fig. 2B, Table S1) composed of short-, medium-, and long-chain fatty acids, many more than were detected using GC/MS (Table S1). Direct profiling of male D. melanogaster’s body (Fig. 2C Upper) and pea aphid’s (Acyrthosiphon pisum) wing (Fig. 2C Lower) showed clear ion signals, presumably lipids, thus documenting the potential of our method for tissue profiling/imaging. Clear spectra were also obtained from submi- croliter volumes of human blood (Fig. 2D), thus highlighting the potential of the matrices for clinical diagnostics by direct analysis of biofluids. Overall, our approach has a wide range of applications— high-throughput metabolomic screening, tissue profiling/imaging, direct biofluids analysis—which can be further developed for biomedical studies. Acid–Base Model Was Further Validated by a Combination of MS, NMR, and X-ray Experiments and Supported by Density Functional Quantum Chemical Calculations. To study the effects of matrix structure and Fig. 1. Comparison of mass spectra of 1,8-bis(dimethylamino)naphthalene basicity, additional bases structurally similar to DMAN were se- (DMAN) with conventional matrices; 4-hydroxy-␣-cyanocinnamic acid (␣- lected, and stearic acid (SA) as a representative acid (pKa ϭ 10.15), CHCA) and 2,5-dihydroxybenzoic acid (DHB) when mixed with stearic acid (SA) was analyzed. A strong dependence of the stearate ion abundance in 1:1 molar ratios. Positive mode analysis. (A) Spectra for ␣-CHCA ϩ SA. (B) on the pKb values of the studied bases was observed (Fig. 3). As in Spectra for DHB ϩ SA. (C) Spectra for DMAN ϩ SA. Spectra in A and B are DMAN, no matrix ions were observed for N,N-dimethylaniline dominated by copious matrix ions with no signal for [MϩH]ϩ of SA at m/z 285 (DMA) (Fig. 3B) and aniline (Fig. 3D). The negative-ion TOF mass (transparent blue bars); clearly, none of the 3 matrices shows the protonated ϩ spectrum obtained by using 1,8-diaminonaphthalene shows, in analyte. C shows DMAN [MϩH] at m/z 215 resulting from DMAN protonated addition to the expected m/z 283 of stearate, copious matrix cluster with SA. Negative mode analysis: (D) Spectra for ␣-CHCA ϩ SA. (B) Spectra for ϩ ϩ ions obscuring both high- and low-mass regions (Fig. 3C). It seems DHB SA. (F) Spectra for DMAN SA. Spectra in D and E show only copious that the gas-phase basicity of the matrix is important and that the matrix clusters with no signal for the deprotonated stearate ion at m/z 283 (transparent red bar); single ion at m/z 283 corresponding to stearate anion is suitable matrix in the negative-ion mode must not tend toward observed, with no additional matrix peaks (F).