草莓中花青素检测方案(生物质谱)

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MALDI-TOF Mass Spectrometry Imaging (MSI)for Distribution Analysis of Anthocyaninsin Strawberry Fruits Imaging mass spectrometry (MSI) via matrix-assisted laser desorption/ionization(MALDI) TOF MS enables the simultaneous detection and localization of molecularspecies across a broad molecular mass range. In biological samples， diverse classesof biomolecules may be sought， including metabolites， sugars， lipids， peptides，and proteins. Keywords：Food analysis， massspectrometry imaging，MALDI-TOFMS Depending on the sample andthe compound class(es) to betargeted in a given MALDI-TOFMsl study， sample preparationschemes (potentialnecessitiesofstructural reinforcement cofdelicate samples， thickness ofsampleSslicing，，cand optionalwashing steps) may vary， as may the selection of the most suitableMALDI matrix to enable desorp-tion and ionization of the targetclasses. Sensitivity and spatialresolution are key elements tosuccessfulMSI studies.， andmatrix application directly influ-ences the quality and quantityofthe spectra data obtained. Uniformity of coverage on thesample surface， matrix crystalsize， and "extractability" of ana-lytes depend on many variables，including the physicalmeansof application， the selection ofapplication solvent(s)， ifttused.and application temperature. The utility of MSI in food analyseshasbeendemonstrated in theidentificationliand quantitationn oofmetabolites， proteins， and pesticides invarious food matrices， including fruitsand vegetables [1]. In this study， thedistribution of anthocyanin species instrawberries (Fragaria x ananassa) wascharacterized by MSI. Anthocyaninsare polyphenolic compounds presentin strawberries， as in other fruits andvegetables naturally red， purple， orbluetoned atmaturity. Beyondproviding vivid color， anthocyaninshave shown to have antioxidative.anti-inflammatory， and antitumoractivities [2-4]， making this class ofcompounds of interest for nutraceu-tical， skin care， and drug researchand development. Pelargonidin andcyanidin derivatives are the mostrepresentative anthocyanins in straw-berries， but this class of compoundsincludes more than 500 entities [2].The identification， distribution， andrelative quantitation of anthocyaninscan support studies ofnaturalspecies andthe development ofgeneticallyymodifiedvarietiesOotsuch fruits for the monitoring anddevelopment of desirable anthocyanincontent characteristics. In additionto "core" profiling via analysis oflongitudinal and cross-sectional slices，analysis of surface slices includingthe strawberry achenes enables amore complete target evaluation andfurther demonstrates the utility andflexibility of MSI technology. Methods and Materials Preliminary Analysis： Preparation andProfiling of Strawberry Extracts "Tochiotome"strawberry(Fragariaananassa Duch.) fruits werecultivated in the Strawberry ResearchCenter (Tochigi， Japan)， harvested inwinter after ripening， and stored at-80°C until use. Precursor ion， Fragment ion， Anthocyanin species (m/z) [M]+ (m/z) [M]+ 433.1 271 Pelargonidin-3-O-glucoside 449.1 287 Cyanidin-3-O-glucoside 479.1 303 Delphinidin-3-O-glycoside 519.1 271 Pelargonidin-3-O-malonylglucoside 535.1 287 Cyanidin-3-O-malonylglucoside 579.1 271 Pelargonidin-3-O-rutinoside thenhomogenized. Homogenateswere suspended in 50% methanol(Sigma-Aldrich， St. Louis， MO， USA)and kept under continuous shakingat room temperature for 2 hours.The solutions were centrifuged at3000 rpm for 15 minutes， and thesupernatants were collectedasextracts for analysis. Strawberry extracts (1 uL) were mixedwith an equal volume of 40 mg/mLDHB (2，5-Dihydroxybenzoic acid， in70% aqueous methanol containing0.1% FA (formic acid). The mixedsolution was deposited onto an indium tin oxide (ITO)-coated glass slide(Bruker Daltonics， Billerica， MA， USA)and dried before MALDI-MS analysis.MALDI-MS and MS/MS analyses wereperformed using an ultrafleXtremeMALDI time-of-flight (TOF)/TOF MSsystem (Bruker Daltonics). MS datawas collected in positive-ion reflectormode across a m/z range of 100-600at a repetition rate of 1000 Hz andwas externally calibrated. For MS/MSanalysis， the selected precursor ionsand the product ions were obtainedusing collision-induced dissociationMS/MS mode. Sample Preparation of StrawberrySections for MSI Fresh mature fruits were immersedin 2% CMC (carboxymethyl cellulosesodium salt. WakoChemicals)and flash frozen in liquid nitrogen.Subsequently， longitudinal andcross sections， 80 pm thick， wereconsecutively prepared using a CM1860 cryostat (Leica Microsystems，Wetzlar， Germany). Sections includingachenes， the small shells enclosing the seeds on the surface of the fruit，were likewise prepared. The sectionswere then mounted onto ITO-coatedglass slides， which were dried in avacuum desiccator for 20 minutes.The mounted sections were placedin 50 mL conical centrifuge tubescontaining silica gel to dry and werestored (within these tubes) at -80°Cuntil analysis. MALDI matrix was appliedthe strawberrysections usinga Figure 2： MS/MS spectra of identified anthocyanins in strawberry crude extract using MALDI-MS/MS.MS/MS spectra of precursor ions at m/zI 433.1，，B1449.1.，C479.1， 1519.1，535.1，and (F579.1 .TM-Sprayerrsystem (HTX Tech-nologies LLC， Carrboro， NC， USA).A DHB solution， 30 mg/mL in 50%aqueous methanol containing 0.1%FA， was used. The solvent flow rate，nozzle speed， number of passes， andtemperature were set at 0.2 mL/min，1200imm/min， 81 and 85 °C，respectively MSl was performed accordingtoaprevious study/ [5]l withsomemodifications.Datawasacquiredon the Bruker ultrafleXtreme MALDItime-of-flight((TOF)/TOF MSwitha step size of 200 pm. The laserdiameter was set to medium size.Other measurement conditions wereas previously described. Data Analysis For the profiling of the strawberryfruit extracts， the obtained MS andMS/MS spectra were analyzed using. flexAnalysis 3.4 software(Bruker. Daltonics).The sugar moieties ofanthocyanin species were identifiedfrom their neutral losses and previousreports[6] For the MSI studies， the acquiredmass spectra were normalized by totalion current (TIC) using flexlmaging4.0 software (Bruker Daltonics). Thesoftware was also used to createtwo-dimensional ion-density maps. Results and Discussion The mass spectrum ot. the ripestrawberry extract is shown in Figure 1.The anthocyanins in the crude extractwere identified byMALDI-MS/MS，withsix unique speciess clearlydetected Based on previouslyanalyses [6]， a number of anthocyaninprecursorions wereexpected，including both anthocyanidin andsugarrmoieties..The detectedanthocyanins are shown in Table 1，including delphinidin-3-O-glycoside，identified in strawberries for the first Figure 3：MALDI-MSI analysis of anthocyanins in strawberry sections. l Mass spectrum obtained from a section. Optical image of a strawberry section.HRepresentative ion images of m/z 433.1， 519.1， 579.1， 449.1， 535.1， and 479.1. Scale bar=5 mm ( Fi gu r e 4 ： MALD I- M S I an a lysis o f a nthocyan i ns i n an ach ene . Op ti c al i mag e o f an ac he n e s e c t ion . R ep res en tat ive i o n images o f m/ z 4 3 3.1， 51 9 .1 ， 5 7 9.1 ， 4 49. 1 ， 53 5. 1 ， a nd 479. 1 . S c a l e b a r =1 mm ) time. The MS/MS spectra， shownin Figure 2，indicate the neutral lossof sugars in each of the anthocyaninsby the detection of their aglyconeTorms. All peaks corresponding to the antho-cyanins identified in the crude extract(Figure 1) were also observed in thespectra from the MSl data collectedon strawberry slices (Figure 3A). Anoptical image of a longitudinal crosssection of a strawberry is shown inFigure 3B. Red pigment is presentin the skin， cortical， and pith tissues of the fruit， indicating the presenceof anthocyanins， and the detectedanthocyanin species showed differentdistribution patterns within thesections. Pelargonidin species weredistributed in the skin， cortical， andpith tissues (Figure 3C-E)， whereascyanidin and delphinidin species werelocalized in the skin (Figure 3F-H).Their aglycones showed similardistribution patterns to theseglycosides (data not shown). An optical image of an achenesection is shown in Figure 4A. As shown in Figure 4B-F， pelargonidinglycosides and cyanidin glycosideswere detected in the (surface) fruitskin， while delphinidin glycoside wasonly slightly detected (Figure 4G).Cyanidin species were also notablydetected on the outer surface of theachene， which is of interest givenprevious studies indicating that theantioxidant capacity of the achene isconsiderably higher than that of thestrawberry receptacle flesh [7]. Conclusion In this study， anthocyanin compounds were detected and localized within "Tochiotome" strawberry fruitsslices via MALDI-TOF MSI on a Bruker ultraflextreme MS system. Embedding the fresh fruit in CMC prior tofreezing and slicing maintained the form of the delicate flesh. Matrix application using the HTX TechnologiesTM-Sprayer provided a uniform and reproducible distribution of the DHB matrix solution， and the use ofelevated temperature (85C) promoted extraction of targeted analytes from the strawberry slices. A preliminarystudy of extracts prepared from the strawberries confirmed the presence and identities of the anthocyaninsThese anthocyanins include delphinidin-3-O-glycoside， identified in strawberries for the first time.MSI techniques， such as those made by MALDI-TOF MS， can rapidly provide a unique depth and breadth. of molecular spatial distribution information within a sample. This analytical approach can provide significantdetail on target compound production during fruit maturation， under different growth conditions， and in varietalselection screens. Knowledge of the expression and localization of these compounds of interest may direct thedevelopment of "healthier” fruits for direct consumer consumption and to improve yields in the preparationof anthocyanin-rich extracts. Further， this analytical approach may be applied to help assess the absorption，distribution， and metabolization of anthocyanin compounds - with both topical and oral administration - as theirpotential benefits for health and well-being are better understood. Learn More You are looking for further Information?Check out the link or scan the QR code for more details. www.bruker.com/maldi-imaging References [1] YoshimuraY， Goto-Inoue N， Moriyama T， Zaima N. 2016. Significant advancement of mass spectrometry imaging for food chemistry. Food Chem， 210， 200-211. [21Lin BW， Gong CC， Song HF， Cui YY. 2016. Effects of anthocyanins on the prevention and treatmentof cancer. Br J Pharmacol， 174(11)， 1226-12433. [3]Giampieri F Gasparrini M， Forbes-HernandezTY， et al. 2018. Overexpression of the AnthocyanidinSynthase Gene in Strawberry Enhances Antioxidant Capacity and Cytotoxic Effects on Human. Hepatic Cancer Cells. J. Agric. Food Chem， 66， 581-592. [4]Nile S H， Park S W. 2014. Edible berries： bioactive components and their effect on human health. Nutrition， 30， 134-144. [5]Zaima N， Sasaki T， Tanaka H， et al. 2011. Imaging mass spectrometry-based histopathologic examination of atherosclerotic lesions.Atherosclerosis， 117， 427-32. [6]Giampieri F Alvarez-Suarez J M， Battino M. 2014. Strawberry and human health： Effects beyondantioxidant activity. J. Agric. Food Chem， 62， 3867-3876 [71Ariza MT， Reboredo-Rodriguez P Mazzoni L，et al. 2016. Strawberry Achenes Are an ImportantSource of Bioactive Compounds for Human Health. Int J Mol Sci.17(7)：1103. For Research Use Only. Not for Use in Clinical Diagnostic Procedures. Bruker Daltonik GmbH Bruker Scientific LLC Bremen· GermanyBillerica， MA· USAPhone +49(0)421-2205-0Phone +1 (978) 663-3660 Author：Hirofumi Enomotoepartment of Biosciences， Faculty of Science and Engineering，Division of Integrated Science and Engineering， Graduate School ofScience and Engineering， and Advanced Instrumental Analysis Center of Teikyo University， Teikyo University， Utsunomiya， Japan ms.sales.bdal@bruker.com-www.bruker.com MALDI TOF 质谱成像可早广泛的分子质量范围内同时检测和定位分子种类，在生物样品中，多种类别的生物分子可被找到，包括代谢物，糖，脂质，肽段和蛋白质。本实验中，花青素通过布鲁克MALDI TOF 质谱系统在草莓中被快速检测并定位到。详情请下载应用文献。