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Preparation of the Partially Methylated Alditol Acetates Derived from CS Tetrasaccharides Containing Galactose for the Gas Chromatography/Mass Spectrometry Analysis
用于气相色谱/质谱分析的含半乳糖CS四糖衍生的部分甲基化糖醇乙酸酯的制备   

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Abstract

Chondroitin sulfate (CS), a member of the glycosaminoglycan (GAG) family of carbohydrates, is composed of linear, sulfated repeating disaccharide sequences of N-acetyl-D-galactosamine (GalNAc) and glucuronic acid (GlcA). Recently, a keratan sulfate (KS) disaccharide [GlcNAc6S(β1-3)Galactose(β1-]-branched CS-E was identified from the clam species M. chinensis. This protocol details a methodology to analyze the glycosidic linkages of galactose in KS disaccharide-branched CS by GC-MS analysis. A complementary method for the identification and characterization of KS-branched CS in M. chinensis can be found in Higashi et al. (2016).

Keywords: Mactra chinensis(中国蛤蜊), Chondroitin sulfate(硫酸软骨素), Keratan sulfate(硫酸角质素), Partially methylated alditol acetates(部分甲基化糖醇乙酸酯), GC-MS analysis(GC-MS分析)

Background

Gas chromatography/mass spectrometry (GC-MS) analysis of the reaction products of partially methylated alditol acetates (PMAA) derived from the polysaccharides has been shown to represent a powerful tool to investigate the glycosidic linkages. The PMAAs preparation from M. chinensis in this protocol was performed according to the method of Anumula and Tayler (1992) with minor modifications.

Materials and Reagents

  1. Screw-cap tube (AGC techno glass, borosilicate Pyrex glass, 13 mm i.d. x 120 mm)
  2. Glass measuring pipette (0.1, 0.5, 1 and 2 ml)
  3. Pasteur pipette IK-PAS-5P (IWAKI, catalog number: 73-0001 )
  4. Keratan sulfate from Bovine Cornea (SEIKAGAKU, catalog number: 400760 )
  5. Dry tetrasaccharide (100 μg) is composed of ∆UA, N-acetyl-D-galactosamine (4S, 6S), galactose, N-acetyl-D-glucosamine (6S). Briefly, KS branched CS from M. chinensis was treated with chondroitinase ACII, and resulting tetrasaccharide was collected through the fractionation using HPLC with Docosil column. Please see details in Higashi et al. (2016)
  6. Dimethyl sulfoxide, dehydrated (dry DMSO) (Wako Pure Chemical Industries, catalog number: 040-18032 )
  7. Iodomethane (CH3I) (Wako Pure Chemical Industries, catalog number: 139-02662 )
  8. Chloroform
  9. Nitrogen gas (> 99.995%) (Nippon Megacare)
  10. Trifluoroacetic acid (TFA) (Wako Pure Chemical Industries, catalog number: 204-02743 )
  11. Acetic acid (NACALAI TESQUE, catalog number: 00212-43 )
  12. 4-N,N-dimethylaminopyridine (Wako Pure Chemical Industries, catalog number: 042-19212 )
  13. Pyridine (NACALAI TESQUE, catalog number: 29509-25 )
  14. Acetic anhydride (Wako Pure Chemical Industries, catalog number: 011-00276 )
  15. Hexane (NACALAI TESQUE, catalog number: 17935-05 )
  16. Sodium hydroxide (NaOH) (NACALAI TESQUE, catalog number: 31511-05 )
  17. Methanol (NACALAI TESQUE, catalog number: 21915-93 )
  18. Dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, catalog number: 043-07216 )
  19. 0.5 mol/L hydrochloric acid methanolic solution (Wako Pure Chemical Industries, catalog number: 080-07725 )
  20. Sodium tetrahydroborate (NaBH4) (Wako Pure Chemical Industries, catalog number: 192-01472 )
  21. NaOH-DMSO suspension (see Recipes)
  22. 5% (v/v) pyridine in 50% acetonitrile/water (see Recipes)
  23. NaBH4 (5 mg/ml) solution (see Recipes)

Equipment

  1. Test tube mixer (SEIKAGAKU, model: TM-251 )
  2. Centrifuge (KUBOTA, model: Model 5922 )
  3. Sample concentrator (Hangzhou Allsheng Instruments, model: MD200-2 )
  4. Time-of-flight mass spectrometer JMS-T100GCV (JEOL, model: JMS-T100GCV )
  5. ZB-5ms column (0.25 µm film thickness, 0.25 µm i.d. x 30 m) (Phenomenex)
  6. Agilent Technologies 7890A GC system (Agilent Technologies, model: Agilent 7890A GC )

Procedure

The preparation of partially methylated alditol acetates (PMAAs) is depicted in Figure 1. All reagents are added using glass pipettes.


Figure 1. Scheme of the preparation of partially methylated alditol acetates (PMAAs) from polysaccharides having galactose residue. (i) DMSO, NaOH-DMSO suspension, CH3I; (ii) TFA (2.5 M); (iii) 5% pyridine in 50% CH3CN/H2O, NaBH4 in 30% CH3OH containing 0.03 M NaOH; (iv) 4-N,N-dimethylaminopyridine in CH3CN, pyridine, acetic anhydride.

  1. Preparation of partially methylated sugars
    1. Dry tetrasaccharide (100 μg) in a screw-cap tube.
    2. Add 200 μl of dry DMSO and sonicate for 30 min.
    3. Add 200 μl of NaOH-DMSO (see Recipes) and vortex.
    4. Transfer the tube containing the sample onto ice and add 100 μl of iodomethane (CH3I).
    5. Sonicate for 5 min and vortex 3 times.
    6. Add 50 μl of CH3I again, and incubate for 30 min on ice.
    7. Add 1 ml of distilled water and then vortex.
    8. Add 1 ml of chloroform using a glass pipet (1 ml), vortex for 1 min, shake the tube well for 1 min, vortex for 1 min.
    9. Centrifuge sample at 900 x g for 5 min at room temperature.
    10. Remove water fraction (upper layer) using Pasteur pipette (aspiration).
    11. To wash organic layer containing the methylated carbohydrates, add 1 ml of water and shake the tube well for 1 min, then vortex for 30 sec. Centrifuge at 900 x g for 5 min at room temperature and remove the water fraction using Pasteur pipette (aspiration).
    12. Wash the organic layer two times using 1 ml of water.
    13. Evaporate organic fraction (lower layer) for 40 min at 40 °C under a stream of nitrogen using a sample concentrator (Figure 2).
    14. Dissolve sample with 150 μl of 2.5 mol/L TFA. Fill with nitrogen gas and close tightly with a screw cap. Incubate at 100 °C for 4 h.
    15. Evaporate sample for 40 min at 40 °C under a stream of nitrogen using a sample concentrator (Figure 2).
    16. Add 500 μl of 5% pyridine in 50% acetonitrile/water (see Recipes) to the dried, partially methylated sugars.


      Figure 2. Evaporation of sample at 40 °C under a stream of nitrogen using sample concentrator (MD200-2). The spray nozzle was inserted into a glass tube which was placed to aluminum dry bath heating block. Sample was dried at 40 °C under nitrogen gas.

  2. Preparation of partially methylated alditol acetates (PMAAs) from partially methylated sugars
    1. Add 200 μl of 5 mg/ml of NaBH4 solution (see Recipes) into the sample and incubate for 4 h at 37 °C.
    2. Add 200 μl of acetic acid and evaporate sample within 1 h at 40 °C under a stream of nitrogen using a sample concentrator (Figure 2).
    3. Dissolve sample with 1 ml of 0.1% (v/v) MeOH-HCl and evaporate sample within 1 h at 40 °C under a stream of nitrogen (Figure 2). Repeat this step four times.
    4. Add 150 μl of 5 mg/ml of 4-N,N-dimethylaminopyridine, 50 μl of pyridine and 150 μl of acetic anhydride, respectively, and incubate at room temperature for 4 h.
    5. After incubation, add 2 ml of distilled water.
    6. Add 2 ml of chloroform, vortex, centrifuge at 900 x g for 5 min at room temperature and remove water (upper layer).
    7. Add 2 ml of distilled water, vortex, centrifuge at 900 x g for 5 min and remove water (upper layer).
    8. Evaporate sample (organic layer) for 40 min at 40 °C under a stream of nitrogen (Figure 2).
    9. Dissolve sample with 30 μl of hexane.

  3. GC-MS (Agilent Technologies 7890A GC system) analysis of PMAAs
    1. Set electro impact ionization, 70 eV.
    2. Set carrier gas, helium at 1.2 ml/min.
    3. Set split less sample injection.
    4. Set column oven temperature program: 3 min at 100 °C, with an increase at 4 °C/min to 160 °C, 1 min at 160 °C followed by an increase at 0.5 °C/min to 180 °C, and a final increase at 20 °C/min to 260 °C and held for 10 min at 260 °C.
    5. Submit 1 μl of PMAAs in hexane to GC-MS.

Data analysis

Electron ionization (EI) which afford the fragment-masses of small compounds is the most common form of ionization for GC-MS analysis. In general, fragmentation patterns generated by EI are compound dependent. In case of the structural analysis of glycans, electron ionization chromatogram (EIC) of certain fragment ions (m/z 45, 117, 161, 233) are useful for identifying peaks corresponding to PMAAs ( Björndal et al., 1970). When EIC of m/z 233 was monitored, PMAA (1,3,5-tri-O-acetyl 2,4,6-tri-O-methyl-galactitol) from galactose in tetrasaccharide of M. chinensis was detected at 28.68 min (Higashi et al., 2016). However, PMAAs from ∆UA, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine were not observed.

Notes

  1. For isolation of KS branched CS-E from M. chinensis and preparation of tetrasaccharide from KS branched CS-E, please see details in Higashi et al. (2016). In general, quantitation of CS was performed by post-column HPLC through the detection of unsaturated disaccharides obtained by chondroitinase (Chase). Interestingly, unknown peaks were found when CS from M. chinensis was treated with Chase ACII (at high concentrations) but not Chase ABC. Thus, unknown peaks were collected and analyzed by LC-MS/MS and GC-MS. In LC-MS/MS analysis, we found that unknown peaks consisted of tetrasaccharides including ∆UA, N-acetyl-D-galactosamine (4S, 6S), hexose, HexNAc (S). In addition, GC-MS analysis suggested that hexose in tetrasaccharide was galactose. Finally, N-acetyl-D-glucosamine (6S) in tetrasaccharide was suggested by 2D-NMR.
  2. PMAAs from M. chinensis were prepared according to the method of Anumula and Taylor (1992) with minor modifications.

Recipes

  1. NaOH-DMSO suspension (prior preparation)
    1. Combine 0.2 ml of 50% (w/w %) NaOH and 0.4 ml of methanol in a screw-cap tube
    2. Dilute with 6 ml of DMSO, vortex and sonicate for 3-5 min
    3. Centrifuge the fine dispersion of NaOH in DMSO at 900 x g for 10 min at room temperature
    4. Resuspend obtained precipitate with 6 ml of fresh DMSO and centrifuge at 900 x g for 10 min at room temperature. Repeat this step two times
    5. Resuspend obtained precipitate with 6 ml of dry DMSO, dehydrated and centrifuge at 900 x g for 10 min at room temperature. Repeat this step three times
    6. Suspend precipitate with 4 ml of dry DMSO and store at 4 °C
  2. 5% (v/v) pyridine in 50% acetonitrile/water (prior preparation)
    1. Prepare 1.9 ml of 50% acetonitrile with distilled water
    2. Combine 0.1 ml of pyridine and 1.9 ml of 50% acetonitrile/water
  3. NaBH4 (5 mg/ml) in 30% methanol containing 0.03 mol/L of NaOH (prior preparation)
    1. Prepare a solution of 0.03 mol/L NaOH in 30% MeOH
    2. Dissolve NaBH4 with 30% MeOH containing 0.03 mol/L of NaOH

Acknowledgments

We thank Dr. Sayaka Masuko for her help in preparing this manuscript. This protocol was adapted from the original work (Higashi et al., 2016) to provide the detailed procedures. This study was supported in part by the Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sport, Science and Technology of Japan and the Inohana Foundation, Chiba University. The authors declare that there's no conflicts of interest.

References

  1. Anumula, K. R. and Taylor, P. B. (1992). A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates. Anal Biochem 203(1): 101-108.
  2. Björndal, H., Hellerqvist, C. G., Lindberg, B. and Svensson, S. (1970). Gas‐liquid chromatography and mass spectrometry in methylation analysis of polysaccharides. Angew Chem Int Ed Engl 9: 610-619.
  3. Higashi, K., Takeda, K., Mukuno, A., Okamoto, Y., Masuko, S., Linhardt, R. J. and Toida, T. (2016). Identification of keratan sulfate disaccharide at C-3 position of glucuronate of chondroitin sulfate from Mactra chinensis. Biochem J 473(22): 4145-4158.

简介

硫酸软骨素(CS)是碳水化合物的糖胺聚糖(GAG)家族的成员,由N-乙酰基-D-半乳糖胺( GalNAc)和葡萄糖醛酸(GlcA)。 最近,从中华蛤中鉴定出硫酸角质素(KS)二糖[GlcNAc6S(β1-3)半乳糖(β1-) - 支化的CS-E。该方案详细描述了分析方法 通过GC-MS分析在KS二糖 - 分枝CS中的半乳糖的糖苷键。可以在Higashi等人中找到用于鉴定和表征KS-分枝CS的互补方法 (2016)。

【背景】已经显示来自多糖的部分甲基化糖醇乙酸酯(PMAA)的反应产物的气相色谱/质谱(GC-MS)分析代表了研究糖苷键的有力工具。 M的PMAAs准备。 在该方案中,根据Anumula和Tayler(1992)的方法进行小的修改。

关键字:中国蛤蜊, 硫酸软骨素, 硫酸角质素, 部分甲基化糖醇乙酸酯, GC-MS分析

材料和试剂

  1. 螺旋盖管(AGC技术玻璃,硼硅酸硼玻璃,13毫米×120毫米)
  2. 玻璃测量吸管(0.1,0.5,1和2毫升)
  3. 巴斯德吸管IK-PAS-5P(IWAKI,目录号:73-0001)
  4. 来自牛角膜的硫酸角质素(SEIKAGAKU,目录号:400760)
  5. 干四糖(100μg)由ΔUA,N-乙酰-D-半乳糖胺(4S,6S),半乳糖,N-乙酰半乳糖胺乙酰基-D-葡糖胺(6S)。简而言之,将来自M.chinensis的KS分枝CS用软骨素酶ACII处理,并通过使用带有Docosil柱的HPLC进行分馏来收集得到的四糖。请参阅Higashi et。(2016)
    的详细信息
  6. 二甲基亚砜,脱水(干燥的DMSO)(和光纯药工业,目录号:040-18032)
  7. 碘甲烷(CH 3 I)(Wako Pure Chemical Industries,目录号:139-02662)
  8. 氯仿
  9. 氮气(> 99.995%)(Nippon Megacare)
  10. 三氟乙酸(TFA)(Wako Pure Chemical Industries,目录号:204-02743)
  11. 醋酸(NACALAI TESQUE,目录号:00212-43)
  12. N,N'-二甲基氨基吡啶(Wako Pure Chemical Industries,目录号:042-19212)
  13. 吡啶(NACALAI TESQUE,目录号:29509-25)
  14. 乙酸酐(Wako Pure Chemical Industries,目录号:011-00276)
  15. 己烷(NACALAI TESQUE,目录号:17935-05)
  16. 氢氧化钠(NaOH)(NACALAI TESQUE,目录号:31511-05)
  17. 甲醇(NACALAI TESQUE,目录号:21915-93)
  18. 二甲基亚砜(DMSO)(Wako Pure Chemical Industries,目录号:043-07216)
  19. 0.5mol / L盐酸甲醇溶液(和光纯药工,产品目录号:080-07725)
  20. 四氢硼酸钠(NaBH 4)(Wako Pure Chemical Industries,目录号:192-01472)
  21. NaOH-DMSO悬浮液(见食谱)
  22. 在50%乙腈/水中的5%(v / v)吡啶(见配方)
  23. NaBH4(5mg / ml)溶液(参见食谱)

设备

  1. 试管混合器(SEIKRGRKU,型号:TM-251)
  2. 离心机(KUBOTA,型号:5922型)
  3. 样品浓缩器(杭州Allsheng仪器,型号:MD200-2)
  4. 飞行时间质谱仪JMS-T100GCV(JEOL,型号:JMS-T100GCV)
  5. ZB-5ms柱(0.25μm膜厚,0.25μm内径×30μm)(Phenomenex)
  6. Agilent Technologies 7890A GC系统(Agilent Technologies,型号:Agilent 7890A GC)

程序

图1描绘了部分甲基化的糖醇乙酸酯(PMAAs)的制备。使用玻璃移液管加入所有试剂。


图1.由具有半乳糖残基的多糖制备部分甲基化的糖醇乙酸酯(PMAAs)的方案。(i)DMSO,NaOH-DMSO悬浮液,CH 3 I; (ii)TFA(2.5M); (iii)在30%CH 3中的50%CH 3 CN / H 2 O,NaBH 4中的5%吡啶,包含0.03M NaOH; (iv)在CH 3 CN,吡啶,乙酸酐中的4- N,N-二甲基氨基吡啶。

  1. 部分甲基化糖的制备
    1. 干燥四糖(100微克)在螺帽管。
    2. 加200μl无水DMSO,超声30分钟。
    3. 加入200μlNaOH-DMSO(见食谱)并涡旋。
    4. 将含有样品的试管转移到冰上并加入100μl碘甲烷(CH 3 I)。
    5. 超声5分钟,涡旋3次。

    6. 再次加入50μlCH 3,在冰上孵育30分钟
    7. 加入1毫升蒸馏水,然后涡旋。
    8. 用玻璃吸管(1毫升)添加1毫升氯仿,涡旋1分钟,充分振摇管1分钟,涡旋1分钟。
    9. 在室温下900×g离心5分钟。

    10. 使用巴斯德吸管(抽吸)去除水分(上层)。
    11. 为了洗涤含有甲基化碳水化合物的有机层,加入1毫升水,充分振摇管1分钟,然后涡旋30秒。在室温下900×g离心5分钟,用巴氏吸管(抽吸)除去水分。

    12. 用1毫升水洗两次有机层
    13. 使用样品浓缩器(图2),在氮气流下在40℃蒸发有机部分(下层)40分钟。
    14. 用150μl2.5mol / L TFA溶解样品。充满氮气,用螺帽密封。
      在100°C孵育4小时

    15. 使用样品浓缩器在40°C氮气流下蒸发样品40分钟
    16. 在干燥的部分甲基化糖中加入500μl5%吡啶的50%乙腈/水(见食谱)。


      图2.使用样品浓缩器(MD200-2)在氮气流下于40℃下蒸发样品。将喷嘴插入置于铝干浴加热块中的玻璃管中。样品在氮气下于40℃干燥。

  2. 由部分甲基化的糖制备部分甲基化的糖醇乙酸酯(PMAAs)
    1. 加入200微升5毫克/毫升的NaBH4溶液(见配方)到样品中,并在37℃孵育4小时。
    2. 加入200μl乙酸,并使用样品浓缩器在40℃和氮气流下在1小时内蒸发样品(图2)。
    3. 用1ml 0.1%(v / v)MeOH-HCl溶解样品,并在40℃和氮气流下在1小时内蒸发样品(图2)。重复此步骤四次。
    4. 加入150μl5mg / ml的N,N'-二甲基氨基吡啶,50μl吡啶和150μl乙酸酐,室温孵育4小时。
    5. 孵化后,加入2毫升蒸馏水。
    6. 加入2ml氯仿,涡旋,在室温下900×g离心5分钟,除去水(上层)。
    7. 加入2毫升蒸馏水,涡旋,900×g离心5分钟,除去水(上层)。

    8. 在40℃氮气流下蒸发样品(有机层)40分钟(图2)
    9. 用30μl己烷溶解样品。

  3. GC-MS(Agilent Technologies 7890A GC系统)分析PMAA
    1. 设置电子轰击电离,70 eV。
    2. 载气,氦气以1.2毫升/分钟。
    3. 设置分割减少样品注入。
    4. 设置柱温箱温度程序:在100℃下3分钟,以4℃/分钟升高至160℃,在160℃下升高1分钟,然后以0.5℃/分钟升高至180℃,最终以20°C / min升至260°C,并在260°C保持10 min。
    5. 将1μl正己烷中的PMAAs提交至GC-MS。

数据分析

提供小化合物片段质量的电子电离(EI)是用于GC-MS分析的最常见的电离形式。一般来说,由EI生成的碎片模式是复合依赖的。在聚糖的某些碎片离子的结构分析,电子电离色谱图(EIC)的情况下( M / Z 45,117,161,233)是用于识别对应于PMAAs(Björndal - 甲基 - 半乳糖醇)从半乳糖的四糖中。在28.68分钟检测到中国猕猴(Higashiet al。,2016)。然而,从PMAAsΔUA,名词的乙酰基<子> d -galactosamine和名词的乙酰基<子> d - 葡糖胺没有被观察到。

笔记

  1. 对于从中华分离的M.chinensis分离KS分支的CS-E和从KS分支的CS-E制备四糖,请参见Higashi等人的详细描述(2016)。通常,CS的定量通过柱后HPLC通过检测由软骨素酶(Chase)获得的不饱和二糖进行。有趣的是,当来自中华稻蝗的CS用高浓度的Chase ACII处理而不是追赶ABC时,发现了未知峰。因此,收集未知峰并通过LC-MS / MS和GC-MS进行分析。在LC-MS / MS的分析,我们发现,未知峰由四糖,包括ΔUA,名词的乙酰基<子> d -galactosamine(4S,6S),己糖,HexNAc (S)。另外,GC-MS分析表明四糖中的己糖是半乳糖。最后,用二维核磁共振证实了四糖中的N-乙酰基 - D-葡糖胺(6S)。
  2. 来自 M的PMAA。
    根据Anumula和Taylor(1992)的方法进行微小的修改

食谱

  1. NaOH-DMSO悬浮液(事先准备)
    1. 将0.2ml的50%(w / w%)的NaOH和0.4ml的甲醇在螺帽管中合并
    2. 用6ml DMSO稀释,涡旋并超声处理3-5分钟
    3. 在室温下,900×g离心10分钟,将DMSO在DMSO中的细分散液在室温下离心。
    4. 用6ml新鲜DMSO重悬得到的沉淀物,并在室温下900×g离心10分钟。重复这个步骤两次
    5. 用6ml无水DMSO重悬所得的沉淀物,脱水并在室温下900×g离心10分钟。重复这个步骤三次
    6. 用4毫升无水二甲基亚砜悬浮沉淀,并储存在4°C
  2. 在50%乙腈/水中的5%(v / v)吡啶(事先制备)

    1. 用蒸馏水准备1.9 ml的50%乙腈
    2. 结合0.1ml吡啶和1.9ml 50%乙腈/水
  3. NaBH 4(5mg / ml)在含有0.03mol / L NaOH的30%甲醇中的溶液(事先制备)
    1. 准备0.03 mol / L的氢氧化钠在30%甲醇中的溶液
    2. 用含有0.03mol / L NaOH的30%MeOH溶解NaBH 4。

致谢

我们感谢Sayaka Masuko博士在准备这份手稿方面的帮助。这个协议是从原来的工作(Higashi et。,2016年)改编,提供详细的程序。这项研究得到了日本文部科学省和日本千叶大学的Inohana基金会的科学研究资助。

参考

  1. Anumula,K.R。和Taylor,P.B。(1992)。 一种从糖蛋白碳水化合物制备部分甲基化糖醇乙酸酯的综合方法 Anal Biochem 203(1):101-108。
  2. Björndal,H.,Hellerqvist,C.G.,Lindberg,B。和Svensson,S.(1970)。 多糖甲基化分析的气液色谱和质谱法 Angew Chem Int Ed Engl 9:610-619。
  3. Higashi,K.,Takeda,K.,Mukuno,A.,Okamoto,Y.,Masuko,S.,Linhardt,R.J。和Toida,T。(2016)。 鉴别来自Mactra chinensis的硫酸软骨素葡糖醛酸的C-3位的硫酸角质素二糖。 Biochem J 473(22):4145-4158。
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引用:Higashi, K. and Toida, T. (2017). Preparation of the Partially Methylated Alditol Acetates Derived from CS Tetrasaccharides Containing Galactose for the Gas Chromatography/Mass Spectrometry Analysis. Bio-protocol 7(21): e2600. DOI: 10.21769/BioProtoc.2600.
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