Metabolite profiling of plastidial deoxyxylulose-5-phosphate pathway intermediates by liquid chromatography and mass spectrometry

Abstract

Metabolite profiling is a powerful tool that enhances our understanding of complex regulatory processes and extends to the comparative analysis of plant gene function. However, at present, there are relatively few examples of metabolite profiling being used to characterize the regulatory aspects of the plastidial deoxyxylulose-5-phosphate (DXP) pathway in plants. Since the DXP pathway is one of two pathways in plants that are essential for isoprenoid biosynthesis, it is imperative that robust analytical methods be employed for the characterization of this metabolic pathway. Recently, liquid chromatography-mass spectrometry (LC-MS), in conjunction with traditional molecular biology approaches, established that the DXP pathway metabolite, methylerythritol cyclodiphosphate (MEcPP), previously known solely as an intermediate in the isoprenoid biosynthetic pathway, is a stress sensor that communicates environmental perturbations sensed by plastids to the nucleus, a process referred to as retrograde signaling. In this chapter, we describe two LC-MS methods from this study that can be broadly used to characterize DXP pathway intermediates.

Fig. 1

Overview of the DXP pathway. Intermediates of the DXP pathway are as follows: Pyruvate (pyr) and glyceraldehyde 3-phosphate (G3P) are condensed to DXP with a loss of carbon dioxide, a reaction performed by DXP synthase (DXS); DXP is reduced to methylerythritol phosphate (MEP) by DXP reductoisomerase (DXR); MEP reacts with cytidine triphosphate to produce diphosphocytidylyl methylerythritol (CDP-ME), a reaction catalyzed by MEP cytidylyltransferase (MCT); CDP-ME is phosphorylated to CDP-ME phosphate (CDP-MEP) by CDP-ME kinase (CMK); CDP-MEP is converted to MEcPP via a loss of cytidine monophosphate, a reaction catalyzed by MEcPP synthase (MDS); MEcPP is converted to hydroxymethylbutenyl diphosphate (HMBPP) by HMBPP synthase (HDS); HMBPP is converted to IPP and DMAPP by HMBPP reductase (HDR); IPP can be reversibly isomerized to DMAPP via IPP isomerase (IDI). MEcPP is thought to undergo reduction and elimination to HMBPP [4]

Fig. 2

LC-ESI-quad MS. After analyte ions are delivered to the ion source, ESI facilitates their transfer (from the liquid to the gas phase) to the entrance of the MS. From there they are transmitted to the quadrupole (via a glass capillary, a skimmer, and focusing lenses) and transit this mass analyzer in a stable spiral like trajectory at its center (while dc and ac/rf voltages are alternated), en route to the detector. This illustration is based on an Agilent Technologies LC-ESI-quad MS system

Fig. 3

LC-ESI-TOF MS. After analyte ions are delivered to the ion source, ESI facilitates their transfer (from the liquid to the gas phase) to the entrance of the MS. From there they are transmitted to the TOF (via a glass capillary, a skimmer, an ion guide and focusing lenses) and are pulsed up the flight tube and reflected down (by specific voltages) en route to the detector. Lighter ions arrive at the detector first. This illustration is based on an Agilent Technologies LC-ESI-quad MS system

Fig. 4

Sample preparation method A. (1) Grind the frozen plant tissue, (2) weigh out 50 mg of plant tissue, (3) add 500 μL of 13 mM ammonium acetate solution and mix thoroughly, (4) centrifuge and transfer the supernatant to a separate centrifuge tube, (5) repeat steps 3 and 4 twice and combine the three extracts, (6) freeze, freeze dry, then reconstitute the dried extract in 100 μL acetonitrile–water (1:1, v/v)

Fig. 5

Sample preparation method B. (1) Flash freeze the plant tissue in liquid nitrogen, (2) transfer the plant tissue to a centrifuge tube containing the pre-chilled grinding balls, (3) transfer the centrifuge tube to a pre-chilled centrifuge tube holder in a liquid nitrogen bath, (4) place the centrifuge tube holder in the mixing chamber of the ball mill and start the grinding cycle, (5) weigh out 50 mg of the plant tissue, (6) add 500 μL of 13 mM ammonium acetate solution and mix thoroughly, (7) centrifuge and transfer the supernatant to a separate centrifuge tube, (8) repeat steps 6 and 7 twice and combine the three extracts, (9) freeze, freeze dry, then reconstitute the dried extract in 100 μL acetonitrile–water (1:1, v/v)

Fig. 6

IXC-IEC-ESI-TOF MS analysis of DXP pathway intermediates. Counts versus time (min). The data was acquired from 80 to 605 m/z

Fig. 7

HILIC-ESI-quad MS analysis of DXP pathway intermediates. Counts versus time (min). The data was acquired in the SIM mode with pre-selected ions of 87, 169, 213, 215, 261, 277, and 520 m/z