- Email: [email protected]
Profiling hydroxycinnamoyl-coenzyme A thioesters: Unlocking the back door of phenylpropanoid metabolism
Analytical Biochemistry 420 (2012) 182–184
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Profiling hydroxycinnamoyl-coenzyme A thioesters: Unlocking the back door of phenylpropanoid metabolism Anthony V. Qualley a, Bruce R. Cooper b, Natalia Dudareva a,⇑ a b
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA Bindley Bioscience Center, Metabolite Profiling Facility, Purdue University, West Lafayette, IN 47907, USA
a r t i c l e
i n f o
Article history: Received 8 June 2011 Received in revised form 9 September 2011 Accepted 10 September 2011 Available online 16 September 2011 Keywords: Hydroxycinnamoyl-CoA esters Plants Acyl-CoA esters Metabolite profiling MS/MS
a b s t r a c t In plants, 20 to 30% of photosynthetically fixed carbon is directed toward lignin and other phenylpropanoid compounds for which hydroxycinnamoyl-coenzyme A (CoA) esters are key intermediates. CoA thioesters, ubiquitous metabolites found in all living cells (often at trace levels), have traditionally been challenging to measure. Here we report a hydrophilic interaction liquid chromatography (HILIC) method, coupled with tandem mass spectrometry (MS/MS), that allows simultaneous sensitive quantification of previously undetectable hydroxycinnamoyl-CoA esters and an extended range of acyl-CoAs from plant tissues. This method provides rapid liquid chromatography (LC) analysis (10 min/sample) and the ability for qualitative assessment of acyl-CoAs by MS/MS precursor ion scanning. Ó 2011 Elsevier Inc. All rights reserved.
All organisms produce metabolically diverse populations of carboxylic acids that are often attached to coenzyme A (CoA)1 forming thioesters, which segregates them from some metabolic fates and directs them toward others. Some cellular processes in which CoA esters participate include fatty acid and branched-chain amino acid metabolism, tricarboxylic acid cycle, and cytosolic isoprenoid biosynthesis [1,2]. In plants, CoA esters are also found in hormone metabolism [3–5]. In addition, hydroxycinnamic acid (hydroxycinnamoyl)-CoA esters are used by plants as intermediates in the biosyntheses of lignin and lignans, flavonoids and isoflavonoids, stilbenes, coumarins, aurones, cutin, suberin, catechin, phenylpropanoid esters, phenylpropenes, and benzenoids [6], which provide structural support to plant cell walls, protect tissues from ultraviolet (UV) radiation, participate in herbivore and pathogen defense, and serve as volatile airborne signals in pollinator attraction [6]. Chemodiversity of plant extracts, as well as labile nature and low abundance of CoA thioesters, has made extraction and quantitation of acyl-CoAs difficult. Liquid chromatography (LC) with UV detection lacks selectivity and sensitivity to measure these compounds without lengthy separations and high-salt mobile phases ⇑ Corresponding author. Fax: +1 765 494 0391. E-mail address: [email protected] (N. Dudareva). Abbreviations used: CoA, coenzyme A; UV, ultraviolet; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ESI, electrospray ionization; HILIC, hydrophilic interaction liquid chromatography; PIS, precursor ion scan; TCA, trichloroacetic acid; SPE, solid-phase extraction; MRM, multiple reaction monitoring; DL, detection limit; QL, quantitation limit; S/N, signal-to-noise; GC, gas chromatography. 1
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.09.010
[7] that have a negative impact on mass spectrometry (MS) [8]. Larson and Graham [9] described a technique to measure acyl-CoAs in plants using chloroacetaldehyde derivatization followed by fluorometric LC but requiring a lengthy, complicated ion-paired separation and an atypical quaternary pump. A newer method employs reversed-phase LC with an ion-pairing reagent to measure eight short-chain acyl-CoAs in animal tissues with sensitive tandem mass spectrometry (MS/MS) detection [10]. Although it is able to rapidly (7 min) separate most targeted analytes, it has neither been evaluated for its ability to resolve other CoA esters nor been used in more complex plant extracts containing coeluting contaminants that compete with analytes for ionization in the MS and increase background, drastically reducing sensitivity. In addition, although ion suppression due to the ion-pairing reagent is mitigated by the infusion of acetonitrile directly into the electrospray ionization (ESI) during analysis, this requires the use of an additional solvent pump not common in LC systems. More recently, another LC–MS/MS method emerged for quantification of short-chain acyl-CoA esters in plants using a reversed-phase separation [11] but cannot be used for analysis of larger (>6-carbon chain length) or more complex acyl-CoA thioesters. Currently, no published method exists for profiling hydroxycinnamoyl-CoA esters from plant tissues. To enable the elucidation of plant phenylpropanoid metabolism, we have developed a rapid (10-min) analytical method for sensitive quantification of hydroxycinnamoyl-CoA esters from plant tissue using hydrophilic interaction liquid chromatography (HILIC) LC–MS/MS. A variety of standards (n-hexanoyl-CoA,
Notes & Tips / Anal. Biochem. 420 (2012) 182–184
hexadecanoyl-CoA, malonyl-CoA, butyryl-CoA, and acetyl-CoA) in addition to hydroxycinnamoyl-CoAs were evaluated for their elution profiles using this LC method. All tested acyl-CoAs coeluted chromatographically, providing the advantage to detect a wide range of acyl-CoAs within a sample and quantify individual compounds by MS/MS. Reconfiguration of MS/MS for precursor ion scan (PIS) mode using daughter ions common to acyl-CoAs allows detection of unanticipated acyl-CoAs. Ring-labeled [13C6]cinnamic acid and [13C6]benzoic acid were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Benzoyl-CoA, malonyl-CoA, acetyl-CoA, butyryl-CoA, n-hexanoylCoA, and hexadecanoyl-CoA were obtained from Sigma–Aldrich (Milwaukee, WI, USA). All other acyl-CoA esters were synthesized enzymatically and purified as described previously [12]. Synthesized acyl-CoAs were analyzed using an Agilent 6210 MSD time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), and product identities were verified by mass analysis. Synthesized purified acyl-CoAs were quantified relative to commercial benzoyl-CoA by LC diode array, monitoring UV absorbance at 260 nm. Separation was performed on a Gemini C6-Phenyl column (4.6 150 mm, 5 lm, 100 Å, Phenomenex, Torrance, CA, USA) with 10 ll injected. LC mobile phases were as follows: 50 mM potassium phosphate (pH 7.0) (solvent A) and methanol (solvent B). Initial conditions were as follows: 10% B for 5 min, followed by a linear gradient to 60% B over 15 min. The column was partitioned back to 10% B over 5 min and equilibrated for 2 min. For sample preparations, 200 mg of petunia flower petals was ground in liquid nitrogen, transferred while frozen to tubes containing 300 ll of 10% (w/v) trichloroacetic acid (TCA) with internal standards (0.5 nmol of [13C]benzoyl-CoA and 0.35 nmol of [13C]cinnamoyl-CoA), and vortexed. Samples were centrifuged for 10 min at 4 °C, and this extraction was repeated. Combined supernatant fractions were mixed with an equal volume of 8% (w/v) ammonium acetate and centrifuged to pellet remaining debris. Solid-phase extraction (SPE) cartridges (1 cc, 50 mg, C-18 Sep Pak Vac, Waters, Milford, MA, USA) were preconditioned by washing with 2 volumes each of methanol, ultrapure water, and 4% ammonium acetate in 5% TCA. Samples were loaded onto SPE columns and washed with 2 volumes of 4% ammonium acetate. Columns were flushed with air, and the analytes eluted using 200 ll of 80% isopropanol and 20% ethyl acetate. For LC–MS/MS, 10 ll of sample was injected onto a Kinetex HILIC column (4.6 100 mm, 2.6 lm, 100 Å, Phenomenex) maintained at 40 °C and analyzed using an Agilent 1200 series LC instrument coupled to an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies). The solvent system was as follows: 60% 10 mM ammonium acetate and 40% 2-propanol (solvent A) and 75% 2-propanol and 25% ethyl acetate (solvent B). The elution gradient was as follows: 0 to 1 min 85% B, 1 to 3 min linear gradient to 30% B, 3 to 5 min 30% B, 5 to 7 min 100% A, and 7 to 10 min 85% B with linear flow gradient from 1 to 1.5 ml/min, completing the run. Elution of acyl-CoAs occurred between 3.5 and 4.5 min under these conditions. During analysis, column effluent was directed to MS/MS from 2.5 to 5.5 min to minimize instrument contamination while adequately determining baseline noise. The Jetstream ESI source was operated in negative ion mode with nozzle and capillary voltages at 0 and 2000 V, respectively. Nebulizer pressure was 55.0 psi, drying gas (nitrogen) was at 350 °C with a flow rate of 8 L/min, and sheath gas was at 325 °C with a flow rate of 8 L/min. The fragmentor was 190 V for all analytes except p-coumaroyland feruloyl-CoAs, optimized at 140 and 150 V, respectively. Multiple reaction monitoring (MRM) was used for selective detection of coeluting acyl-CoAs, although it resulted in a somewhat irregular peak shape for lower abundance compounds. The first quadrupole was set to transition between the seven ions corresponding to the [M H] of analytes, whereas the last quadrupole monitored m/z
183
408, characteristic of all acyl-CoAs and corresponding to formation of an adenosine phosphate monophosphate fragment after the loss of H2O [11]. Each transition was monitored with a dwell time of 50 ms. Collision energies were 40 V for all analytes except feruloyland caffeoyl-CoAs, optimized at 50 and 45 V, respectively. Ultrapure nitrogen was used as collision gas. Mass selection was done using the following ions (corresponding to [M H] of our analytes of interest): [13C6]cinnamoyl-CoA, 902.1; cinnamoyl-CoA, 896.1; [13C6]benzoyl-CoA, 876.1; benzoyl-CoA, 870.1; p-coumaroyl-CoA, 912.1; caffeoyl-CoA, 928.1; feruloyl-CoA, 942.1. All data were collected and analyzed with MassHunter Workstation (version B.02, Agilent Technologies). Response factors were determined for each hydroxycinnamoyl-CoA ester by analyzing known amounts of authentic compounds spiked with constant amounts of [13C6]cinnamoyl-CoA and [13C6]benzoyl-CoA as internal standards; whichever internal standard displayed a response factor closest to 1 for a given compound was used for quantification. Detector responses for measured acyl-CoA esters were linear and reproducible across tested concentrations (0.05–25 pmol) (Fig. 1A). The detection limit (DL) for each compound was determined based on standard deviations of responses, whereas the quantitation limit (QL) was defined as the amount of analyte injected that produced a signal-to-noise (S/ N) ratio greater than 10 [13]. These values are shown in Fig. 1B in addition to measured amounts of hydroxycinnamoyl-CoAs in petunia petals (n = 3). Recovery of hydroxycinnamoyl-CoA esters from plant tissue, estimated from recovery of internal standards added to plant sample prior to extraction, was 81.8 ± 18.5% (mean ± standard deviation). Recovery was calculated as the ratio between the measured amount of standard after extraction and the known amount of standard only added just prior to analysis of identically prepared samples. Detection and quantitation by MS/MS of endogenous hydroxycinnamoyl-CoA esters in analyzed petunia petals is shown in Fig. 1C. Hydroxycinnamoyl-CoA esters eluted simultaneously at 4 min, as did all tested acyl-CoA standards (observed by UV absorbance at 260 nm). This method was also successfully applied to profile hydroxycinnamoyl-CoA esters from 200 mg of Arabidopsis leaves (data not shown). Due to coelution of acyl-CoAs
Fig.1. LC–MS/MS detection of hydroxycinnamoyl-CoA esters. (A) Standard curve showing a linear response across tested range. Points represent means ± standard deviations (n = 3). (B) Analytical sensitivity for hydroxycinnamoyl-CoA esters. (B and C) Profiling of hydroxycinnamoyl-CoA esters in petunia petal tissue. Traces in panel C are labeled with the compound name, quantity detected, and S/N values.
184
Notes & Tips / Anal. Biochem. 420 (2012) 182–184
sensitivity to current methodologies. The ability to measure hydroxycinnamoyl-CoAs, as well as to perform metabolite profiling of acyl-CoAs, provides new opportunities for investigations in phenylpropanoid metabolism and will result in a more comprehensive understanding of carbon flux through a wide variety of metabolic networks. Acknowledgments This work was supported by a grant from the National Science Foundation (MCB-0919987) to N.D. and by the Bindley Bioscience Center (Purdue University). The authors are grateful to Karl V. Wood for useful discussions and critical reading of the manuscript. Fig.2. Precursor ion scan of petunia petal extract elution peak (m/z from 800 to 950) while monitoring at product ion of m/z 408.
and the inability of MS/MS to discriminate certain isomers, this method is unable to distinguish acyl-CoA esters of equal mass. Attention should be given to acyl-CoAs for which more than one structure is predicted for a given molecular mass. Gas chromatography (GC)/MS analysis of organic acids after saponification of sample [14] will help to determine organic acid potential for formation of CoA esters. To simulate ‘‘blind’’ metabolite profiling and detect unknown acyl-CoA esters in petunia petals, LC was performed as described above using previously characterized samples with the MS/MS operated in PIS mode. The first quadrupole was set to scan a precursor m/z range from 800 to 950, whereas the third quadrupole was fixed to monitor a fragment ion common to all acyl-CoAs (m/z 408). Fig. 2 is a plot of all precursor ions producing detectable signals within the acyl-CoA peak (3.75–4.25 min). Three of five targeted endogenous analytes, as well as both internal standards, were easily observed despite increased background noise and lower sensitivity in PIS mode when compared with MRM. Thus, operation of MS/MS in PIS mode while monitoring for a common product may aid the identification of additional, previously undetected acyl-CoA thioesters when combined with our developed LC method. The method reported here describes a novel, facile, and rapid approach for quantitation of previously undetectable hydroxycinnamoyl-CoA esters in addition to a wide range of other plant acyl-CoAs. The chromatographic coelution of a large number of acyl-CoAs offers (i) qualitative assessment of previously obscured acyl-CoAs in a sample, (ii) a universal approach for profiling targeted acyl-CoAs, and (iii) comparable throughput and
References [1] I.A. Graham, P.J. Eastmond, Pathways of straight and branched chain fatty acid catabolism in higher plants, Prog. Lipid Res. 41 (2002) 156–181. [2] K.A. Lucas, J.R. Filley, J.M. Erb, E.R. Graybill, J.W. Hawes, Peroxisomal metabolism of propionic acid and isobutyric acid in plants, J. Biol. Chem. 282 (2007) 24980–24989. [3] I.F. Acosta, H. Laparra, S.P. Romero, E. Schmelz, M. Hamberg, J.P. Mottinger, M.A. Moreno, S.L. Dellaporta, tasselseed1 is a lipoxygenase affecting jasmonic acid signaling in sex determination of maize, Science 323 (2009) 262–265. [4] B.K. Zolman, B. Bartel, An Arabidopsis indole-3-butyric acid-response mutant defective in PEROXIN6, an apparent ATPase implicated in peroxisomal function, Proc. Natl. Acad. Sci. USA 101 (2004) 1786–1791. [5] C. Li, A.L. Schilmiller, G. Liu, G.I. Lee, S. Jayanty, C. Sageman, J. Vrebalov, J.J. Giovannoni, K. Yagi, Y. Kobayashi, G.A. Howe, Role of b-oxidation in jasmonate biosynthesis and systemic wound signaling in tomato, Plant Cell 17 (2005) 971–986. [6] T. Vogt, Phenylpropanoid biosynthesis, Mol. Plant 3 (2010) 2–20. [7] A.W. Tumaney, J.B. Ohlrogge, M. Pollard, Acetyl coenzyme A concentrations in plant tissues, J. Plant Phys. 161 (2004) 485–488. [8] J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong, C.M. Whitehouse, Electrospray ionization: principles and practice, Mass Spectrom. Rev. 9 (1990) 37–70. [9] T.R. Larson, I.A. Graham, A novel technique for the sensitive quantification of acyl CoA esters from plant tissues, Plant J. 25 (2001) 115–125. [10] L. Gao, W. Chiou, H. Tang, X. Cheng, H.S. Camp, D.J. Burns, Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS, J. Chromatogr. B 853 (2007) 303–313. [11] M.A. Perera, S.Y. Choi, E.S. Wurtele, B.J. Nikolau, Quantitative analysis of shortchain acyl-coenzyme A in plant tissues by LC–MS/MS electrospray ionization method, J. Chromatogr. B 877 (2009) 482–488. [12] T. Beuerle, E. Pichersky, Enzymatic synthesis and purification of aromatic coenzyme A esters, Anal. Biochem. 302 (2002) 305–312. [13] ICH Harmonised Tripartite Guideline Validation of Analytical Procedures: Text and Methodology, Q2(R1), Current Step 4 Version, Parent Guideline on Methodology, International Conference on Harmonisation, Geneva, Switzerland, 2005. [14] I. Orlova, A. Marshall-Colon, J. Schnepp, B. Wood, M. Varbanova, E. Fridman, J.J. Blakeslee, W.A. Peer, A.S. Murphy, D. Rhodes, E. Pichersky, N. Dudareva, Reduction of benzenoid synthesis in petunia flowers reveals multiple pathways to benzoic acid and enhancement in auxin transport, Plant Cell 18 (2006) 3458–3475.
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Profiling hydroxycinnamoyl-coenzyme A thioesters: Unlocking the back door of phenylpropanoid metabolism Anthony V. Qualley a, Bruce R. Cooper b, Natalia Dudareva a,⇑ a b
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA Bindley Bioscience Center, Metabolite Profiling Facility, Purdue University, West Lafayette, IN 47907, USA
a r t i c l e
i n f o
Article history: Received 8 June 2011 Received in revised form 9 September 2011 Accepted 10 September 2011 Available online 16 September 2011 Keywords: Hydroxycinnamoyl-CoA esters Plants Acyl-CoA esters Metabolite profiling MS/MS
a b s t r a c t In plants, 20 to 30% of photosynthetically fixed carbon is directed toward lignin and other phenylpropanoid compounds for which hydroxycinnamoyl-coenzyme A (CoA) esters are key intermediates. CoA thioesters, ubiquitous metabolites found in all living cells (often at trace levels), have traditionally been challenging to measure. Here we report a hydrophilic interaction liquid chromatography (HILIC) method, coupled with tandem mass spectrometry (MS/MS), that allows simultaneous sensitive quantification of previously undetectable hydroxycinnamoyl-CoA esters and an extended range of acyl-CoAs from plant tissues. This method provides rapid liquid chromatography (LC) analysis (10 min/sample) and the ability for qualitative assessment of acyl-CoAs by MS/MS precursor ion scanning. Ó 2011 Elsevier Inc. All rights reserved.
All organisms produce metabolically diverse populations of carboxylic acids that are often attached to coenzyme A (CoA)1 forming thioesters, which segregates them from some metabolic fates and directs them toward others. Some cellular processes in which CoA esters participate include fatty acid and branched-chain amino acid metabolism, tricarboxylic acid cycle, and cytosolic isoprenoid biosynthesis [1,2]. In plants, CoA esters are also found in hormone metabolism [3–5]. In addition, hydroxycinnamic acid (hydroxycinnamoyl)-CoA esters are used by plants as intermediates in the biosyntheses of lignin and lignans, flavonoids and isoflavonoids, stilbenes, coumarins, aurones, cutin, suberin, catechin, phenylpropanoid esters, phenylpropenes, and benzenoids [6], which provide structural support to plant cell walls, protect tissues from ultraviolet (UV) radiation, participate in herbivore and pathogen defense, and serve as volatile airborne signals in pollinator attraction [6]. Chemodiversity of plant extracts, as well as labile nature and low abundance of CoA thioesters, has made extraction and quantitation of acyl-CoAs difficult. Liquid chromatography (LC) with UV detection lacks selectivity and sensitivity to measure these compounds without lengthy separations and high-salt mobile phases ⇑ Corresponding author. Fax: +1 765 494 0391. E-mail address: [email protected] (N. Dudareva). Abbreviations used: CoA, coenzyme A; UV, ultraviolet; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; ESI, electrospray ionization; HILIC, hydrophilic interaction liquid chromatography; PIS, precursor ion scan; TCA, trichloroacetic acid; SPE, solid-phase extraction; MRM, multiple reaction monitoring; DL, detection limit; QL, quantitation limit; S/N, signal-to-noise; GC, gas chromatography. 1
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.09.010
[7] that have a negative impact on mass spectrometry (MS) [8]. Larson and Graham [9] described a technique to measure acyl-CoAs in plants using chloroacetaldehyde derivatization followed by fluorometric LC but requiring a lengthy, complicated ion-paired separation and an atypical quaternary pump. A newer method employs reversed-phase LC with an ion-pairing reagent to measure eight short-chain acyl-CoAs in animal tissues with sensitive tandem mass spectrometry (MS/MS) detection [10]. Although it is able to rapidly (7 min) separate most targeted analytes, it has neither been evaluated for its ability to resolve other CoA esters nor been used in more complex plant extracts containing coeluting contaminants that compete with analytes for ionization in the MS and increase background, drastically reducing sensitivity. In addition, although ion suppression due to the ion-pairing reagent is mitigated by the infusion of acetonitrile directly into the electrospray ionization (ESI) during analysis, this requires the use of an additional solvent pump not common in LC systems. More recently, another LC–MS/MS method emerged for quantification of short-chain acyl-CoA esters in plants using a reversed-phase separation [11] but cannot be used for analysis of larger (>6-carbon chain length) or more complex acyl-CoA thioesters. Currently, no published method exists for profiling hydroxycinnamoyl-CoA esters from plant tissues. To enable the elucidation of plant phenylpropanoid metabolism, we have developed a rapid (10-min) analytical method for sensitive quantification of hydroxycinnamoyl-CoA esters from plant tissue using hydrophilic interaction liquid chromatography (HILIC) LC–MS/MS. A variety of standards (n-hexanoyl-CoA,
Notes & Tips / Anal. Biochem. 420 (2012) 182–184
hexadecanoyl-CoA, malonyl-CoA, butyryl-CoA, and acetyl-CoA) in addition to hydroxycinnamoyl-CoAs were evaluated for their elution profiles using this LC method. All tested acyl-CoAs coeluted chromatographically, providing the advantage to detect a wide range of acyl-CoAs within a sample and quantify individual compounds by MS/MS. Reconfiguration of MS/MS for precursor ion scan (PIS) mode using daughter ions common to acyl-CoAs allows detection of unanticipated acyl-CoAs. Ring-labeled [13C6]cinnamic acid and [13C6]benzoic acid were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Benzoyl-CoA, malonyl-CoA, acetyl-CoA, butyryl-CoA, n-hexanoylCoA, and hexadecanoyl-CoA were obtained from Sigma–Aldrich (Milwaukee, WI, USA). All other acyl-CoA esters were synthesized enzymatically and purified as described previously [12]. Synthesized acyl-CoAs were analyzed using an Agilent 6210 MSD time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), and product identities were verified by mass analysis. Synthesized purified acyl-CoAs were quantified relative to commercial benzoyl-CoA by LC diode array, monitoring UV absorbance at 260 nm. Separation was performed on a Gemini C6-Phenyl column (4.6 150 mm, 5 lm, 100 Å, Phenomenex, Torrance, CA, USA) with 10 ll injected. LC mobile phases were as follows: 50 mM potassium phosphate (pH 7.0) (solvent A) and methanol (solvent B). Initial conditions were as follows: 10% B for 5 min, followed by a linear gradient to 60% B over 15 min. The column was partitioned back to 10% B over 5 min and equilibrated for 2 min. For sample preparations, 200 mg of petunia flower petals was ground in liquid nitrogen, transferred while frozen to tubes containing 300 ll of 10% (w/v) trichloroacetic acid (TCA) with internal standards (0.5 nmol of [13C]benzoyl-CoA and 0.35 nmol of [13C]cinnamoyl-CoA), and vortexed. Samples were centrifuged for 10 min at 4 °C, and this extraction was repeated. Combined supernatant fractions were mixed with an equal volume of 8% (w/v) ammonium acetate and centrifuged to pellet remaining debris. Solid-phase extraction (SPE) cartridges (1 cc, 50 mg, C-18 Sep Pak Vac, Waters, Milford, MA, USA) were preconditioned by washing with 2 volumes each of methanol, ultrapure water, and 4% ammonium acetate in 5% TCA. Samples were loaded onto SPE columns and washed with 2 volumes of 4% ammonium acetate. Columns were flushed with air, and the analytes eluted using 200 ll of 80% isopropanol and 20% ethyl acetate. For LC–MS/MS, 10 ll of sample was injected onto a Kinetex HILIC column (4.6 100 mm, 2.6 lm, 100 Å, Phenomenex) maintained at 40 °C and analyzed using an Agilent 1200 series LC instrument coupled to an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies). The solvent system was as follows: 60% 10 mM ammonium acetate and 40% 2-propanol (solvent A) and 75% 2-propanol and 25% ethyl acetate (solvent B). The elution gradient was as follows: 0 to 1 min 85% B, 1 to 3 min linear gradient to 30% B, 3 to 5 min 30% B, 5 to 7 min 100% A, and 7 to 10 min 85% B with linear flow gradient from 1 to 1.5 ml/min, completing the run. Elution of acyl-CoAs occurred between 3.5 and 4.5 min under these conditions. During analysis, column effluent was directed to MS/MS from 2.5 to 5.5 min to minimize instrument contamination while adequately determining baseline noise. The Jetstream ESI source was operated in negative ion mode with nozzle and capillary voltages at 0 and 2000 V, respectively. Nebulizer pressure was 55.0 psi, drying gas (nitrogen) was at 350 °C with a flow rate of 8 L/min, and sheath gas was at 325 °C with a flow rate of 8 L/min. The fragmentor was 190 V for all analytes except p-coumaroyland feruloyl-CoAs, optimized at 140 and 150 V, respectively. Multiple reaction monitoring (MRM) was used for selective detection of coeluting acyl-CoAs, although it resulted in a somewhat irregular peak shape for lower abundance compounds. The first quadrupole was set to transition between the seven ions corresponding to the [M H] of analytes, whereas the last quadrupole monitored m/z
183
408, characteristic of all acyl-CoAs and corresponding to formation of an adenosine phosphate monophosphate fragment after the loss of H2O [11]. Each transition was monitored with a dwell time of 50 ms. Collision energies were 40 V for all analytes except feruloyland caffeoyl-CoAs, optimized at 50 and 45 V, respectively. Ultrapure nitrogen was used as collision gas. Mass selection was done using the following ions (corresponding to [M H] of our analytes of interest): [13C6]cinnamoyl-CoA, 902.1; cinnamoyl-CoA, 896.1; [13C6]benzoyl-CoA, 876.1; benzoyl-CoA, 870.1; p-coumaroyl-CoA, 912.1; caffeoyl-CoA, 928.1; feruloyl-CoA, 942.1. All data were collected and analyzed with MassHunter Workstation (version B.02, Agilent Technologies). Response factors were determined for each hydroxycinnamoyl-CoA ester by analyzing known amounts of authentic compounds spiked with constant amounts of [13C6]cinnamoyl-CoA and [13C6]benzoyl-CoA as internal standards; whichever internal standard displayed a response factor closest to 1 for a given compound was used for quantification. Detector responses for measured acyl-CoA esters were linear and reproducible across tested concentrations (0.05–25 pmol) (Fig. 1A). The detection limit (DL) for each compound was determined based on standard deviations of responses, whereas the quantitation limit (QL) was defined as the amount of analyte injected that produced a signal-to-noise (S/ N) ratio greater than 10 [13]. These values are shown in Fig. 1B in addition to measured amounts of hydroxycinnamoyl-CoAs in petunia petals (n = 3). Recovery of hydroxycinnamoyl-CoA esters from plant tissue, estimated from recovery of internal standards added to plant sample prior to extraction, was 81.8 ± 18.5% (mean ± standard deviation). Recovery was calculated as the ratio between the measured amount of standard after extraction and the known amount of standard only added just prior to analysis of identically prepared samples. Detection and quantitation by MS/MS of endogenous hydroxycinnamoyl-CoA esters in analyzed petunia petals is shown in Fig. 1C. Hydroxycinnamoyl-CoA esters eluted simultaneously at 4 min, as did all tested acyl-CoA standards (observed by UV absorbance at 260 nm). This method was also successfully applied to profile hydroxycinnamoyl-CoA esters from 200 mg of Arabidopsis leaves (data not shown). Due to coelution of acyl-CoAs
Fig.1. LC–MS/MS detection of hydroxycinnamoyl-CoA esters. (A) Standard curve showing a linear response across tested range. Points represent means ± standard deviations (n = 3). (B) Analytical sensitivity for hydroxycinnamoyl-CoA esters. (B and C) Profiling of hydroxycinnamoyl-CoA esters in petunia petal tissue. Traces in panel C are labeled with the compound name, quantity detected, and S/N values.
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Notes & Tips / Anal. Biochem. 420 (2012) 182–184
sensitivity to current methodologies. The ability to measure hydroxycinnamoyl-CoAs, as well as to perform metabolite profiling of acyl-CoAs, provides new opportunities for investigations in phenylpropanoid metabolism and will result in a more comprehensive understanding of carbon flux through a wide variety of metabolic networks. Acknowledgments This work was supported by a grant from the National Science Foundation (MCB-0919987) to N.D. and by the Bindley Bioscience Center (Purdue University). The authors are grateful to Karl V. Wood for useful discussions and critical reading of the manuscript. Fig.2. Precursor ion scan of petunia petal extract elution peak (m/z from 800 to 950) while monitoring at product ion of m/z 408.
and the inability of MS/MS to discriminate certain isomers, this method is unable to distinguish acyl-CoA esters of equal mass. Attention should be given to acyl-CoAs for which more than one structure is predicted for a given molecular mass. Gas chromatography (GC)/MS analysis of organic acids after saponification of sample [14] will help to determine organic acid potential for formation of CoA esters. To simulate ‘‘blind’’ metabolite profiling and detect unknown acyl-CoA esters in petunia petals, LC was performed as described above using previously characterized samples with the MS/MS operated in PIS mode. The first quadrupole was set to scan a precursor m/z range from 800 to 950, whereas the third quadrupole was fixed to monitor a fragment ion common to all acyl-CoAs (m/z 408). Fig. 2 is a plot of all precursor ions producing detectable signals within the acyl-CoA peak (3.75–4.25 min). Three of five targeted endogenous analytes, as well as both internal standards, were easily observed despite increased background noise and lower sensitivity in PIS mode when compared with MRM. Thus, operation of MS/MS in PIS mode while monitoring for a common product may aid the identification of additional, previously undetected acyl-CoA thioesters when combined with our developed LC method. The method reported here describes a novel, facile, and rapid approach for quantitation of previously undetectable hydroxycinnamoyl-CoA esters in addition to a wide range of other plant acyl-CoAs. The chromatographic coelution of a large number of acyl-CoAs offers (i) qualitative assessment of previously obscured acyl-CoAs in a sample, (ii) a universal approach for profiling targeted acyl-CoAs, and (iii) comparable throughput and
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