This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany&#8217;s Excellence Strategy - EXC-2070 &#8211; 390732324 (PhenoRob), the Research Training Group GRK2064 and individual research grants SCHA1274/4-1 and SCHA1274/5-1 to G.S.. We also thank Li Schl&#252;ter and Brigitte Ueberbach for technical assistance.

The authors have nothing to disclose.

Here we present a versatile and sensitive method to quantify InsPs including PP-InsPs in plant extracts and provide practical tips on how to get this method established. Even though the protocol is generally robust, suboptimal runs and analyses can occur. In most cases, those runs can be identified by a strong reduction or even complete loss of highly phosphorylated InsPs, especially the PP-InsP species InsP7 and InsP8. Possible reasons can be microbial contaminations of the plant material and insufficient deactivation of endogenous plant PP-InsP hydrolases during extraction due to insufficient grinding and thawing of plant material that will not be in immediate contact with extraction buffer. Further reasons include inaccurate pH adjustment by insufficient or excess addition of neutralization buffer, or simply insufficient sample material. The latter can make it difficult to detect PP-InsPs, since those are often present in very low amounts in the cells. An excess of sample material or inefficient drying during step 3.5 may cause dilution of the perchloric acid, therefore also leading to insufficient enzyme deactivation and a specific loss of InsP6 and PP-InsPs. The amount of plant material, as well as radiolabel used in this protocol were optimized based on costs and performance, and is therefore close to the lowest amount that is still sufficient for providing optimal results. In addition, the column resin will gradually loose its resolution&#160;capacity. The first sign of this process is (for reasons not entirely clear to the authors) a specific loss of higher phosphorylated InsP species like the PP-InsPs in the HPLC spectrum. With further aging, even InsP6 will not be resolved properly by the column (Figure 1D). Therefore, the use of an adequate column, as well as meticulous handling of the sample and proper maintenance of the HPLC components is crucial for ensuring accurate results.
When comparing samples and runs, especially when generated with different equipment (e.g., HPLC systems and columns) or on different days, it is crucial to normalize the samples to each other (as described in step 7.3) and to analyze them in the same way. Only through normalization it is possible and accurate to show multiple samples in the same graph (Figure 3). For quantification of individual InsPs relative to total InsPs, or to another specific InsP species, it is not necessary to normalize, as long as only relative values and not absolute values are shown. Ideally, both the InsP profiles and the quantifications are shown. However, in some cases it is not possible to adequately show two or more runs in the same graph. Different retention times or different levels of background activity can make it difficult to compare unquantified SAX-HPLC profiles alone. The same is the true when many samples need to be compared. In such cases, a further evaluation using an additional software (e.g., Origin) for individual peak quantification is necessary.
The authors are aware that the protocol described here can be optimized and needs to be adapted to each individual research question. Although being optimized for Arabidopsis extracts7,17 in this protocol, this method is versatile and can help determining InsP profiles of other plant species as well. Here we exemplify this possibility by presenting for the first time a InsP profile for L. japonicus, which required no modifications of the labeling conditions, InsP extraction or SAX-HPLC run (Figure 2). Notably, while overall similar, differences are observed between L. japonicus and Arabidopsis InsP profiles. For instance, in L. japonicus InsP5 [4-OH] or its enantiomeric form InsP5 [6-OH] are more abundant than InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH] in comparison to Arabidopsis, where InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH] are the dominant InsP5 species. Likewise, we anticipate that alterations in the media composition, [3H]-myo-inositol concentration, plant age, environmental conditions (e.g., light and temperature), addition of chemical compounds or analyses of plant-microbial interactions among other factors, might need to be tested and adapted.
One important drawback of this method that needs to be considered is that the labeling is done in a (sterile) liquid culture, which does not represent a physiological environment for most land plants. In addition, due to the high costs of [&#179;H]-myo-inositol, the volume of the labeling solution and the size of the culture vessel is generally limited, which also restricts the size of the plants that can be used. Cultivation in liquid culture can be avoided by directly infiltrating for instance leaves of soil-grown plants with [&#179;H]-myo-inositol and subsequently following the protocol described here, as previously reported10.
There are several drawbacks of this protocol in comparison to alternative methods, such as TiO2 pull-down followed by PAGE or mass spectrometry based techniques. Due to the [3H]-myo-inositol labeling, only InsP species that directly originate from radiolabeled myo-inositol will be detected in the end. The method described here is blind to other Ins isomers such as scyllo-inositol and other isomers some of which have been identified in certain plants44. Furthermore, myo-InsPs derived from other pathways will be excluded, including those synthesized by de novo synthesis of myo-inositol and myo-inositol-3-phosphate via isomerization of glucose-6-phosphate, catalyzed by myo-inositol-3-phosphate synthase (MIPS) proteins45. Although [32P] or [33P]-ortho-phosphate can be used as alternative labels, their use poses a major disadvantage, since every phosphate-containing molecule, including the abundant nucleotides and its derivatives, will be labeled. Those molecules can also be extracted with this protocol and bind to the SAX column, which will result in a high level of background activity that will interfere with the identification of individual InsP peaks5. In addition, quantification of [32P]- or [33P] -labeled InsPs and PP-InsPs can be strongly influenced by phosphate and pyrophosphate moiety turnover and might not report a mass readout for inositol species.
On the other hand, [3H]-myo-inositol specifically labels myo-inositol-containing molecules. InsPs, inositol-containing lipids, such as phosphoinositides, and galactinol are in this case labeled. However, only InsPs will be analyzed with this protocol, since lipids are insoluble in the extraction buffer and galactinol does not bind to the SAX&#160;column.
So far, the differences from a plant InsP profile generated by [3H]-myo-inositol labeling compared with one determined by TiO2 pulldown/PAGE remains unknown, since such comparisons have not been performed in plants. A recent study in animal cells addressed this question46. In that work, a pool of InsP6 that is invisible by [3H]-myo-inositol labeling, which should thereby be directly derived from glucose-6-phosphate, was identified by comparing SAX-HPLC profiles with PAGE gels of mammalian cell lines. 24 h of phosphate starvation resulted in a 150% increase of InsP6 when quantifying PAGE gels of InsPs purified using TiO2 pulldown. SAX-HPLC analyses of [3H]-myo-inositol-labeled cells that were treated identically only showed an increase by 15% of [3H]-InsP6. As previously mentioned, InsPs lower than InsP5 are undetectable with PAGE analysis in most cases. Radiolabeling followed by SAX-HPLC appears to be the method of choice, as long as mass spectrometric protocols are not optimized to detect this group of highly negatively charged molecules.
Another remaining challenge is to distinguish enantiomers in SAX-HPLC analyses (or in any other method for InsP analysis)10,17. This challenge can be tackled by the addition of chiral selectors, i.e., enantiopure compounds like L-arginine amide that interact with the respective enantiomeric molecules to form diastereomeric complexes that can be separated10. To our knowledge, this approach has only been implemented to discriminate the enantiomeric InsP5 isomers InsP5&#160;[1-OH] and InsP5 [3-OH] by NMR analyses10. Discrimination of other enantiomeric pairs or successful discrimination of enantiomers by chiral SAX-HPLC analysis or chiral PAGE-based methods have not yet been reported and should be further developed. Considering the conserved synthesis and the conserved regulation of PP-InsPs by phosphorous availability, we envision that especially non-radioactive methods such as PAGE or MS-based methods, together with nutrient analyses, will help ground truthing efforts to calibrate remote sensing data designed to diagnose nutrient deficiencies in crops17,18,24,25. However, the method presented here can currently still be considered the gold standard for InsP analyses and will be instrumental to discover new functions of these intriguing messengers in plants.

Almost four decades ago, inositol phosphates (InsPs) emerged as signaling molecules, after Ins(1,4,5)P3 (InsP3) was identified as a second messenger that activates the receptor-mediated release of Ca2+ in animal cells1,2. To date, no InsP3 receptor (IP3-R) has been identified in plants, which questions a direct signaling role for InsP3 in plant cells3. Regardless, InsP3 serves as a precursor for other InsPs involved in several plant developmental processes, including the regulation of specific signaling pathways3,4,5,6,7,8. For instance, InsP3 can be further phosphorylated to InsP6, also known as &#8220;phytic acid&#8221;, which represents a major source of phosphate, myo-inositol and cations, and was shown to play key roles in plant defense against pathogens, mRNA export and phosphate homeostasis5,9,10,11,12.
Inositol pyrophosphates (PP-InsPs) are a class of InsPs that contain at least one high-energy di-phospho bond, initially identified in animal cells, amoeba and yeast, where they play critical roles in various cellular processes13,14,15. Despite seminal work on PP-InsPs in plants16,17,18,19,20,21,22,23,24,25,26, the biological functions and isomer identity of these molecules still remain largely enigmatic. In the model plant Arabidopsis thaliana, cellular InsP8 was proposed to regulate defenses against insect herbivores and necrotrophic fungi via coincidence-detection of InsP8 and active jasmonate by the ASK1-COI1-JAZ receptor complex17. Furthermore, roles of InsP8 and other PP-InsPs in energy homeostasis and nutrient sensing, as well as phosphate homeostasis have been proposed17,23,24,25,26.
Regardless of the biological system employed, one major methodological challenge when studying InsPs has been the reliable detection and precise quantification of these molecules. Mass spectrometry-based methods have been used to detect InsPs, including PP-InsPs, from cell extracts.&#160;However,&#160;those studies failed to differentiate distinct isomers26,27. Another approach to analyze InsPs employs pull-down of InsPs from cell lysates using TiO2 beads, followed by polyacrylamide gel electrophoresis (PAGE) of the eluted InsPs. The InsPs can then be stained by either toluidine blue or DAPI24,28,29. However, it is so far not possible to reliably detect InsPs lower than InsP5 from plant extracts using this method. Recently, a method using [13C]-myo-inositol for nuclear magnetic resonance (NMR) analysis of InsPs was published as an alternative to strong anion exchange high-performance liquid chromatography (SAX-HPLC)30. This technique has been reported to achieve a similar sensitivity compared to SAX-HPLC and to allow the detection of 5-InsP7, as well as the discrimination of different non-enantiomeric InsP5 isomers from cell extracts. However, the implementation of the NMR-based method&#160;requires chemically synthesized and commercially not available [13C]-myo-inositol. Therefore, the method employed in most cases is radiolabeling samples with [3H]-myo-inositol, followed by SAX-HPLC31,32,33. This technique is based on the uptake of radioactive myo-inositol into the plant and its conversion into different InsPs by the combined activity of dedicated cellular kinases and phosphatases.
The [3H]-labeled InsPs are then acid-extracted and fractionated using SAX-HPLC. Because of their negative charge, the InsPs strongly interact with the positively charged stationary phase of the SAX-HPLC column and can be eluted with a buffer gradient containing increasing phosphate concentrations to outcompete InsPs from the column. Elution times thus depend on charge and geometry of the InsP species to be separated. In the absence of chiral columns, only non-enantiomeric isomers can by separated by this protocol. However, radiolabeled standards can be used to assign the isomeric nature of a specific InsP peak. Multiple efforts in the past by various laboratories to generate labeled and unlabeled standards with (bio)chemical methods or to purify them from various cells and organisms have helped assigning peaks to certain InsP species, and also to narrow down the isomeric identity of individual InsP species5,7,21,34,35,36,37,38,39,40,41,42,43. Also, the recent elucidation of enzymatic pathways leading to the formation of PP-InsPs in plants, as well as the discovery of a bacterial type III effector with a specific 1-phytase activity, provide information on how to generate useful standards for these analyses10,17,18,22,23.
The resulting fractions can be measured in a liquid scintillation counter due to the &#946;-decay of tritium (3H). With increasing labeling time, a steady-state isotopic equilibrium is reached, after which the obtained InsP profiles should represent the InsP status of the plant31. The major advantage of this protocol in comparison to other available techniques is the high sensitivity achieved by the use of the direct precursor for InsPs and the measurement of a radioactive signal.
SAX-HPLC of samples extracted from [3H]-myo-inositol-labeled plants or other organisms is commonly used for the detection and quantification of InsPs ranging from&#160;lower InsP species to&#160;PP-InsPs, representing a valuable tool to better understand the metabolism, function and modes of action of InsPs. So far, this method is also the most appropriate choice for researchers with special interest in lower InsP species. While the basics of this procedure, on which the protocol described here builds on, have been previously described7,21,31,34, a detailed protocol tailored to the analysis of plant-derived InsPs and especially of PP-InsPs is still missing. Previous publications reported difficulties to reliably detect the low abundant PP-InsPs, especially InsP8, due to one or more of the following factors: relatively low amounts of plant material, [3H]-myo-inositol with low specific activity (&#62; 20 Ci/mmol), usage of&#160;extraction buffers that are either not based on perchloric acid or are less concentrated than 1 M, different neutralizing buffers, as well as sub-optimal gradients or detection of [3H] with an on-line detector. In comparison to those studies, the protocol presented here is designed for the reliable detection of PP-InsPs7,21,34.
Here we present a detailed workflow, starting from the setup of the equipment to plant cultivation and labeling, InsP extraction and the SAX-HPLC run itself. Although the method was optimized to the model plant A. thaliana, it can be easily modified to study other plant species, as shown here with the first reported InsP profile of the model legume Lotus japonicus. Although the use of a different plant species might require some optimization, we envisage that those will be minor, making this protocol a good starting point for further research in plant InsPs. In order to facilitate possible optimizations, we indicate every step within the protocol in which modifications are possible, as well as all critical steps that may be challenging when establishing the method for the first time. Additionally, we report how data obtained by this method can be used for the quantification of specific InsPs and how different samples can be analyzed and compared.

<table><tbody><tr><td>Centrifuge</td><td>Eppendorf AG</td><td>model: 5430 R</td><td></td></tr><tr><td>Diammonium hydrogen phosphate, &amp;ge;97 %</td><td>Carl Roth GmbH + Co. KG</td><td>0268.1</td><td></td></tr><tr><td>Ethylenediamine tetraacetic acid disodium salt dihydrate, &amp;ge;99 %, p.a., ACS</td><td>Carl Roth GmbH + Co. KG</td><td>8043.2</td><td></td></tr><tr><td>Falcon 12-well clear flat bottom TC-treated multiwell cell culture plate, with lid, individually wrapped, sterile</td><td>Corning Inc.</td><td>353043</td><td></td></tr><tr><td>Fraction collector</td><td>LAMBDA Instruments GmbH</td><td>model: OMNICOLL single channel collector</td><td></td></tr><tr><td>Growth incubator</td><td>poly klima GmbH</td><td>model: PK 520-LED</td><td></td></tr><tr><td>HPLC pumps</td><td>Kontron Instruments</td><td>model: 420</td><td></td></tr><tr><td>HPLC syringe for Rheodyne valves, 1 mL</td><td>Hamilton Company</td><td>81365</td><td></td></tr><tr><td>Injector for HPLC</td><td>Supelco</td><td>model: Rheodyne 9725</td><td></td></tr><tr><td>Inositol, myo-[1,2-3H(N)]</td><td>American Radiolabeled Chemicals Inc.</td><td>ART 0261</td><td></td></tr><tr><td>Liquid nitrogen</td><td>University, Chemistry Department</td><td></td><td></td></tr><tr><td>Liquid scintillation counter</td><td>PerkinElmer Inc</td><td>model: TRI-CARB 2900TR</td><td></td></tr><tr><td>Micro pestle</td><td>Carl Roth GmbH + Co. KG</td><td>CXH7.1</td><td></td></tr><tr><td>Mixed cellulose Eester filter, ME range (ME 24), plain, 0.2 &amp;micro;m pore size, 47 mm circle</td><td>GE Healthcare Life Sciences</td><td>10401770</td><td></td></tr><tr><td>Mixer for HPLC</td><td>Kontron Instruments</td><td>model: M 800</td><td></td></tr><tr><td>Murashige &amp;amp; Skoog medium, salt mixture</td><td>Duchefa Biochemie</td><td>M0221</td><td></td></tr><tr><td>OriginPro software</td><td>OriginLab Corp.</td><td></td><td></td></tr><tr><td>Orthophosphoric acid, &amp;ge;85 %, p.a., ISO</td><td>Carl Roth GmbH + Co. KG</td><td>6366.1</td><td></td></tr><tr><td>Partisphere 5 &amp;micro;m SAX cartridge column, 125 x 4.6 mm</td><td>Hichrom Limited</td><td>4621-0505</td><td></td></tr><tr><td>Perchloric acid, 70 %, 99.999 % trace metals basis</td><td>Sigma-Aldrich</td><td>311421</td><td></td></tr><tr><td>Petri dish, square, PS, clear, 120/120/17 mm, sterile</td><td>Greiner Bio One International GmbH</td><td>688161</td><td></td></tr><tr><td>pH-indicator paper pH 5.5 - 9.0, Neutralit</td><td>Merck KGaA</td><td>109564</td><td></td></tr><tr><td>Phytagel</td><td>Sigma-Aldrich</td><td>P8169</td><td></td></tr><tr><td>Potassium carbonate</td><td>Carl Roth GmbH + Co. KG</td><td>P743.2</td><td></td></tr><tr><td>Safe-Lock tubes, 1.5 mL</td><td>Eppendorf AG</td><td>30120086</td><td></td></tr><tr><td>Sample loop for 9725 injectors, volume 2 mL, PEEK</td><td>Supelco</td><td>57648</td><td></td></tr><tr><td>SNAPTWIST scintillation vial, 6.5 mL</td><td>Simport Scientific Inc.</td><td>S207-5</td><td></td></tr><tr><td>Sterile bench</td><td>LaboGene</td><td>model: ScanLaf MARS 900</td><td></td></tr><tr><td>Sucrose, &amp;ge;99,5 %, p.a.</td><td>Carl Roth GmbH + Co. KG</td><td>4621.1</td><td></td></tr><tr><td>Ultima-Flo AP liquid scintillation cocktail</td><td>PerkinElmer Inc</td><td>6013599</td><td></td></tr><tr><td>Ultra-pure deionized water</td><td>Milli-Q</td><td></td><td></td></tr><tr><td>Wrenchless WVS End Fitting Kit</td><td>Hichrom Limited</td><td>4631-1001</td><td></td></tr></tbody></table>

extraction and quantification of soluble, radiolabeled inositol polyphosphates from different plant species using sax-hplc

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

The results shown here aim to illustrate possible outcomes obtained according to variations at technical and biological levels. The first is exemplified by analyses using new versus aged columns (Figure 1) and fresh versus stored samples (Figure 3), and the second by evaluating extracts from two different plant systems, A. thaliana (Figure 1, Figure 3) and L. japonicus (Figure 2).
An optimal SAX-HPLC run is depicted on Figure 1A&#8210;C, which shows a complete inositol polyphosphate spectrum obtained from A. thaliana extracts after scintillation counting. Note that peaks are nicely separated and can be assigned to different isomers (or enantiomer-pairs) based on chromatographic mobilities described earlier5,7.
Figure 2 shows the representative result of a SAX-HPLC analysis of L. japonicus seedlings that were grown and labeled under the same conditions as the Arabidopsis seedlings. While presumably all InsP species and peaks that are known from Arabidopsis can be seen, there are substantial differences regarding the relative (e.g., ratios between isomers) amount of specific InsP isomers, when comparing the profiles of both species. For instance, the Lotus extracts showed increased InsP3c, InsP4b, InsP5b and reduced InsP3a, InsP4a, InsP5a and InsP5c compared to Arabidopsis which leaves room for further investigations. Figure 2D illustrates the different ratios between InsP isomers between Arabidopsis and Lotus.
Figure 3 shows two InsP profiles of a sample that was split after the extraction. The first half was immediately analyzed and the second half one day later, after storage at -80 &#176;C. Note that only minor&#160; differences are observed between the different samples (i.e., black and red lines on Figure 3A&#8210;C, and Figure 3D). This illustrates that one freeze-thaw cycle does not harm the sample and that the method itself generates reproducible results.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61495/61495fig01.jpg" /> 
Figure 1: Typical InsP profile of a successful and of an unsuccessful SAX-HPLC analysis performed with this protocol.&#160;(A&#8210;C) SAX-HPLC profile of 17-day-old wild-type (Col-0) Arabidopsis seedlings radiolabeled with [3H]-myo-inositol. Global InsP extraction and SAX-HPLC run were performed on the same day. (A) Full spectra; (B, C) Zoom-ins of the profile shown in A. All visible peaks are highlighted and assigned to the corresponding InsP species. Based on published chromatographic mobilities5,7, InsP4a likely represents Ins(1,4,5,6)P4 or Ins(3,4,5,6)P4, InsP5a represents InsP5 [2-OH], InsP5b represents InsP5 [4-OH] or its enantiomeric form InsP5 [6-OH], and InsP5c represents InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH]. The isomeric natures of InsP3a-c, InsP4b, InsP7, and InsP8 are still unknown. Panel (D) shows a SAX-HPLC profile of identically grown plants but using an aged column (&#62;40 runs). A clear reduction of InsP6 compared to other InsP species and the absence of PP-InsPs is visible. <a href="https://www.jove.com/files/ftp_upload/61495/61495fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61495/61495fig2v2.jpg" /> 
Figure 2: Representative InsP profile of L. japonicus plants.&#160;SAX-HPLC profile (A&#8210;C) of 17-day-old wild-type (Gifu) L. japonicus seedlings radiolabeled with [3H]-myo-inositol. (A) Full spectra; (B, C) Zoom-ins of the profile shown in A. All visible peaks are highlighted and assigned to the corresponding InsP species. Based on published chromatographic mobilities5,7, InsP4a likely represents Ins(1,4,5,6)P4 or Ins(3,4,5,6)P4, InsP5b likely represents InsP5 [4-OH] or its enantiomeric form InsP5 [6-OH], and InsP5c likely represents InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH]. The isomeric natures of InsP3a-c, InsP4b, InsP7, and InsP8 are unknown. (D) Comparison between the individual InsP species (in % of total activity from elution 25&#8210;96) of A. thaliana (data from Figure 1A&#8210;C) and L. japonicus (data from Figure 2A&#8211;C). <a href="https://www.jove.com/files/ftp_upload/61495/61495fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61495/61495fig03.jpg" /> 
Figure 3: InsP profiles of a split sample illustrating the reproducibility of SAX-HPLC analyses.&#160;(A&#8210;C) SAX-HPLC profiles of 17-day-old wild-type (Col-0) Arabidopsis seedlings radiolabeled with [3H]-myo-inositol. Prior the run, the sample was split and one half run immediately and the other half one day later after storage at -80 &#176;C. (A) Full spectra; (B, C) Zoom-ins of the profile shown in A. All visible peaks are highlighted and assigned to the corresponding InsP species. Based on published chromatographic mobilities5,7, InsP4a likely represents Ins(1,4,5,6)P4 or Ins(3,4,5,6)P4, InsP5a represents InsP5 [2-OH], InsP5a represents InsP5 [2-OH], InsP5b represents InsP5 [4-OH] or its enantiomeric form InsP5 [6-OH], and InsP5c represents InsP5 [1-OH] or its enantiomeric form InsP5 [3-OH]. The isomeric natures of InsP3a-c, InsP4b, InsP7, and InsP8 are still unknown. Panel D shows the quantification of InsP6 and the PP-InsPs InsP7 and InsP8 of both runs. The values represent the amount (in %) of the respective InsP species relative to all InsP (total activity from elution 25&#8211;96). <a href="https://www.jove.com/files/ftp_upload/61495/61495fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Software settings for liquid scintillation counting using a light scintillation counter. Screenshots showing the software version, as well as settings used for scintillation counting of [3H] samples performed with this protocol are depicted. <a href="https://cloudflare.jove.com/files/ftp_upload/61495/61495supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplemental Figure 2: Representative example of data normalization. A screenshot of a worksheet shows all steps and formulas used to normalize SAX-HPLC runs to each other. For simplification, only fractions 25&#8211;35 of samples are shown. <a href="https://cloudflare.jove.com/files/ftp_upload/61495/61495supfig02.jpg" target="_blank">Please click here to download this figure.</a>
Supplemental Figure 3: Peak determination, background subtraction and integration using analysis software. (A) Data from SAX-HPLC analysis is loaded into the software (minutes 28&#8211;96) and the peak analyzer tool is selected. (B&#8210;E) The baseline is defined manually by setting points between individual peaks and the background is subtracted. (F) Peaks are determined manually based on appearance and published chromatographic mobilities5,7. (G) Peak ranges are defined manually by cpm values. (H) Peaks are integrated and calculated as % of all peaks. <a href="https://cloudflare.jove.com/files/ftp_upload/61495/61495supfig03.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC at JoVE.com

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

extraction-quantification-soluble-radiolabeled-inositol

Research

JoVE Journal

Biochemistry

6.9K Views.  University of Bonn. Het hier aangetoonde protocol kan helpen om de biosynthese van inositolpolyfosfaten van planten te verduidelijken en vragen te beantwoorden over hun rol in bepaalde aspecten van plantenmetabolisme en -ontwikkeling. Deze methode is zeer gevoelig omdat het gebruik van de radioactief gelabelde precursor van inositolfosfaten een betrouwbare detectie van alle inositolfosfaatsoorten in planten mogelijk maakt. Visuele demonstratie van deze methode is belangrijk omdat de meeste plantenbiologen niet b...

Video: Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Environment

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Bioengineering

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Chemistry

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography.

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.

A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Biology

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

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