Determination of Mn Concentrations in Synechocystis sp. PCC6803 Using ICP-MS

引用 收藏 提问与回复 分享您的反馈 Cited by



Plant Physiology
Mar 2017



Manganese (Mn) is an essential micronutrient for all photoautotrophic organisms. Two distinct pools of Mn have been identified in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), with 80% of the Mn residing in the periplasm and 20% in cytoplasm and thylakoid lumen (Keren et al., 2002). In this protocol, we describe a method to quantify the periplasmic and intracellular pools of Mn in Synechocystis accurately, using inductively coupled plasma mass spectrometry (ICP-MS).

Keywords: Cyanobacteria (蓝藻), Synechocystis (集胞藻属), Manganese (锰), Periplasm (细胞周质), ICP-MS (ICP-MS)


Mn plays a vital role in the active sites of several enzymes such as the oxygen-evolving complex in photosystem II. In contrast to its role as an important micronutrient, Mn can be toxic when present in excess. It is therefore of crucial importance for cyanobacteria to maintain the intracellular levels of Mn and in particular to avoid free Mn in the cytoplasm. The cyanobacterium Synechocystis addresses this challenge by storing about 80% of the Mn in the periplasm. Only 20% of the cellular content can be detected in the cytoplasm and thylakoid system (Keren et al., 2002), with most of the Mn being incorporated into proteins, leaving virtually no free Mn in the cytoplasm. We recently identified the manganese transport protein Mnx, which resides in the thylakoid membrane and facilitates export of Mn from the cytoplasm into the thylakoid lumen in Synechocystis. According to our study, Mnx is a major player in maintaining the cellular Mn homeostasis (Brandenburg et al., 2017). To analyze the biological significance of Mnx, we developed a protocol to measure the periplasmic and intracellular Mn pools separately. We chose ICP-MS for quantification, since it is a sensitive and reliable method to detect metals in biological samples. Detection limits can be in the range of [ng L-1] and below. Prior to the analysis, all complex molecules of the sample are broken down to atomic compounds by digestion with nitric acid. Subsequently, the sample is ionized by an inductively coupled plasma and analyzed by mass spectrometry. In this protocol, we describe the detailed workflow for subcellular Mn quantification, from sampling to the calculation of Mn concentrations.

Materials and Reagents

  1. 15 ml tubes (SARSTEDT, catalog number: 62.554.002 )
  2. Syringe filter filtropur S, 0.2 µm (SARSTEDT, catalog number: 83.1826.001 )
  3. 10 ml Luer-Lok syringe without needle (BD, catalog number: 300912 )
  4. Milli-Q grade (or similar) water (Resistivity at 25 °C 18 MΩ cm)
  5. Distilled AR grade nitric acid (Avantor Performance Materials, MACRON, catalog number: 1409-46 )
  6. ICP-MS multi element standard solution VI (Merck, catalog number: 1105800100 )
  7. Na2-Ethylenediaminetetraacetic acid (EDTA) (Carl Roth, catalog number: 8043.2 )
  8. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Carl Roth, catalog number: 6763.3 )
  9. EDTA wash buffer (see Recipes)


  1. Cooling centrifuge for 15 ml tubes (Beckman Coulter, model: GS-6 )
  2. Teflon cups (Savillex, custom made)
  3. Analytical balance (KERN & SOHN, model: ALJ 220-4M )
  4. Vortex Mixer (Eppendorf, model: MixMate® )
  5. Clean lab Class 5000 (according to US FED STD 209E, which would be in between ISO6 and ISO 7 according to the newer ISO 14644-1 and ISO 14698 standards)
  6. Chemical hood (Wesemann)
  7. Hot plate (Ceramic Stirring Hot Plate) (IKA, catalog number: 35810.01 )
  8. ICP-MS 7500cx (Agilent Technologies, model: ICP-MS 7500cx )
  9. Neubauer-improved hemocytometer (Marienfeld-Superior, catalog number: 0650030 )


  1. Sampling and washing (Figure 1)
    1. Keep samples on ice to slow down the metabolic activity.
    2. The washing steps are time sensitive since they stop the experiment by removing all external Mn. Removing all external Mn conserves the sample in the state of the timepoint it was taken and the elemental composition of the sample cannot change anymore.
    3. Sample volumes and final elution volumes are accounted for in the calculation under data analysis. All volumes in this protocol are therefore approximate volumes, as long as they are weighted accurately. All transfers can be done by pouring. Pipetting is not necessary and may even add contaminants.

      Figure 1. Workflow for sampling and washing. A. Intracellular (sample C) and periplasmic (samples E1 and E2) Mn pools are separated by EDTA washing steps. B. To measure the total cellular Mn content (sample T), cells are separated from the medium and resuspended in Milli-Q water.

    1. For each sample, prepare four 15 ml test tubes, labeled with sample number and T1 (total cell), E1 (first EDTA wash), E2 (second EDTA wash) and C1 (intracellular Mn content), respectively.
    2. Weigh and note the weight of each test tube, including cap.
    3. Take 2 ml samples (OD750 ~0.75, Note 3) and transfer them into tubes T1 and C1 (Note 4).
    4. Remove 30 µl from tube T1 for cell counting.
    5. Weigh and note the weight of tubes T1 and C1.
      Note: Subtracting the weight of the empty tube from that of the tube with sample gives you the weight of the sample.
    6. Centrifuge tubes T1 and C1 at 3,000 x g and 4 °C for 5 min.
    7. Discard the supernatant.
      Note: The supernatant can be analyzed as sample M (medium) if wanted. If so, weigh the sample before filtering it through a 0.2 µm syringe filter to get rid of remaining cells. The growth of remaining cells would change the Mn composition of the medium and require sample digestion. After filtering, the sample does not need further processing.
    8. Resuspend samples T1 by vortexing in approximately 4 ml of Milli-Q water. Store the samples at room temperature until sample digestion.
    9. Add 4 ml EDTA wash buffer to samples C and resuspend by vortexing. Repeat step 1f.
    10. Transfer the EDTA supernatant to the test tube labeled E1. Weigh and note the weight. Store tube E1 at room temperature. The sample does not need further processing (Note 5).
      Note: Most ICP-MS facilities do not accept undigested or unfiltered samples. Therefore, it may be necessary to filter the EDTA washes as described above. However, potentially remaining cells cannot grow, since they are in EDTA wash buffer and not medium.
    11. Add 4 ml EDTA wash buffer to samples C and resuspend by vortexing. Repeat step 1f.
    12. Transfer the EDTA supernatant to the test tube labeled E2. Weigh and note the weight. Store tube E2 at room temperature. The sample does not need further processing.
    13. Add 4 ml Milli-Q water and resuspend by vortexing.
    14. Remove 30 µl from the tube for cell counting.
  2. Sample digestion
    Note: From here on, it is essential to carry out all steps in clean room conditions.
    1. For each sample, prepare another set of two 15 ml test tubes (label with sample number and C2 or T2).
    2. Weigh out 4 ml of Milli-Q water to the test tubes C2 and T2
    3. Add 2 ml of nitric acid to each of samples T1 and C1.
    4. Gently shake or invert for 5 min.
    5. Preheat a hot plate in a chemical hood to 200 °C (Note 6).
    6. Place each sample (T1 and C1 only) into Teflon cups suitable for the hot plate and place the samples on the hot plate.
    7. Let the samples evaporate until minimal droplet remains; remove from the hot plate before sample evaporates completely (Note 7).
    8. Add all water from corresponding test tube to the Teflon cup, re-suspend the sample carefully and return the sample to the same test tube (e.g., water from tube T2 to the evaporated sample T and back to tube T2).
  3. Sample measurement (clean room not required)
    1. Transfer 2 ml from each sample (T2, C2, E1, and E2) to appropriate sample tubes for the ICP-MS sample handler.
    2. Store the remaining 2 ml of each sample for potential extra measurements.
    3. Analyze the samples according to the instructions of your ICP-MS facility.
  4. Cell counting
    1. Perform cell counting on the samples from sampling and washing (steps 1d and 1n) according to the instructions manual of your hemocytometer.

Data analysis

The periplasmic Mn concentration is the sum of the Mn in samples E1 and E2. Sample C2 is the intracellular concentration of Mn, namely cytosol and thylakoid system. Apply equation 1 to calculate the periplasmic Mn concentration using samples E1, E2 as well as volume and cell count from C1. Similarly, use equation 2 to calculate the intracellular Mn concentration in [atoms cell-1] using samples C1 and C2.

ICP-MS results usually come in parts per million (ppm), which is equivalent to [µg ml-1] or parts per billion (ppb), which is equivalent to [µg L-1]. Make sure to convert the input data into the correct units. Please note that the molecular weight (MW) in equations 1 and 2 is in [µg mol-1].
Instead of cell counting, you can use OD750 or chlorophyll content for normalization as well. However, we recommend cell counting. As a control, the sum of the Mn in samples C2, E1 and E2 should equal the Mn concentration in sample T2. You can calculate sample T2 in the same way as sample C2.
Figure 2 shows an example of the calculations. The numbers used for this calculation were obtained from a WT sample grown at our standard conditions (Note 1).

Figure 2. Example calculation of Mn concentration. Make sure the input data is in the correct units. In our case, for example, the ICP-MS results were in [µg L-1]. Please note that the molecular weight (MW) of Mn is in [µg mol-1]. In our experiments, the difference between E1 + E2 + C2 and T2 was in the range of 0.1-10 x 103 [atoms cell-1].


  1. Mutant lines are based on and compared to a Japanese WT of Synechocystis sp. PCC 6803 obtained from Martin Hagemann (University of Rostock, Germany). Our standard growth conditions are 30 °C, 100 rpm shaking, and 100 µmol photons m-2 sec-1.
  2. This is an elemental analysis that does not differentiate between Mn incorporated into proteins, complexed Mn and free Mn.
  3. The amount of original sample to be analyzed depends on the cell density of the sample. Dense samples require less volume and dilute samples require more. There is no need to adjust the other volumes. In our experiments, we took 2 ml samples with an OD750 ~0.75.
  4. There is no need to use a pipette following this protocol. This minimizes the contamination of the sample. All transfers from one tube to another should be done by pouring.
  5. In principle, it is possible to combine samples E1 and E2 and measure them together. If doing so, the sample volume in the calculation needs then to be changed accordingly. However, depending on the physiological conditions of the experiment, the amount of Mn found in these samples can be close to the detection limit already in sample E1. Therefore, we prefer to not further dilute sample E1 with sample E2.
  6. The temperature of the hot plate can be set as low as 180 °C if handling a large number of samples. Doing this will slow down the process allowing for better tracking of all samples on the hot plate to avoid burning.
  7. The minimal possible droplet size mostly depends on the experience of the person conducting the experiment. Importantly, the droplet size gets included in the sample weight before the measurements meaning larger droplets do not affect the measurements. This step is solely to protect the ICP-MS pump by reducing the amount of nitric acid in the sample. If samples do evaporate completely, add 1 ml of nitric acid and mix well. Continue evaporation carefully.
  8. All samples can be stored at room temperature until digestion, as soon as the experiment has stopped by the washing steppes. All possible contaminations will be eliminated in the digestion process but cannot change the elemental composition of the sample. We are not aware of any specific shelf life of the samples before digestion, however, the longest time we stored samples before digestion was three weeks. After digestion, we recommend freezing the samples to prevent microbial growth which may affect the ICP-MS machine rather than the sample composition.


  1. EDTA wash buffer
    20 mM HEPES-KOH, pH 7.5
    5 mM EDTA
    Note: EDTA wash buffer can be stored at room temperature for several months.


This research was supported by the German Science Foundation (EI 945/3-1 to M.E.) and the Israeli Science Foundation (2733/16 to N.K.). The authors declare that there are no conflicts of interest.


  1. Brandenburg, F., Schoffman, H., Kurz, S., Kramer, U., Keren, N., Weber, A. P. and Eisenhut, M. (2017). The Synechocystis manganese exporter Mnx is essential for manganese homeostasis in cyanobacteria. Plant Physiol 173(3): 1798-1810.
  2. Keren, N., Kidd, M. J., Penner-Hahn, J. E. and Pakrasi, H. B. (2002). A light-dependent mechanism for massive accumulation of manganese in the photosynthetic bacterium Synechocystis sp. PCC 6803. Biochemistry 41(50): 15085-15092.


锰(Mn)是所有光合自养生物体必需的微量营养素。 在蓝细菌集胞藻中已经鉴定了两个不同的锰库。 PCC 6803( Synechocystis ),其中80%的Mn存在于周质中,20%存在于细胞质和类囊体腔中(Keren等,2002)。 在这个协议中,我们描述了使用电感耦合等离子体质谱法(ICP-MS)精确定量集胞藻中周质和细胞内锰库的方法。

【背景】Mn在几种酶的活性位点中起着至关重要的作用,例如光系统II中的放氧复合物。与其作为重要的微量营养素的作用相反,锰过量时可能是有毒的。因此,蓝细菌维持细胞内的Mn水平,特别是避免游离的Mn在细胞质中是至关重要的。蓝细菌集胞藻通过在周质中储存约80%的Mn来解决这一挑战。在细胞质和类囊体系统中只能检测到20%的细胞含量(Keren等,2002),大部分Mn被掺入蛋白质中,在细胞质中几乎没有游离的Mn 。我们最近鉴定了锰转运蛋白Mnx,它存在于类囊体膜中并促进Mn从细胞质输出到集胞藻的类囊体腔中。根据我们的研究,Mnx是维持细胞Mn稳态的主要参与者(Brandenburg等人,2017)。为了分析Mnx的生物学意义,我们制定了一个方案来分别测量周质和细胞内Mn池。我们选择ICP-MS进行定量分析,因为它是检测生物样品中金属的灵敏可靠的方法。检测限可以在[ng L ]和以下的范围内。在分析之前,样品的所有复杂分子都通过用硝酸消化分解成原子化合物。随后,通过电感耦合等离子体将样品离子化并通过质谱分析。在这个协议中,我们描述了从采样到锰浓度计算的亚细胞锰定量的详细工作流程。

关键字:蓝藻, 集胞藻属, 锰, 细胞周质, ICP-MS


  1. 15毫升管(SARSTEDT,目录号:62.554.002)
  2. 注射器过滤器filtropur S,0.2μm(SARSTEDT,目录号:83.1826.001)
  3. 10毫升Luer-Lok无针注射器(BD,目录号:300912)
  4. Milli-Q级(或类似)水(电阻率25°C,18MΩcm)
  5. 蒸馏AR级硝酸(Avantor Performance Materials,MACRON,目录号:1409-46)
  6. ICP-MS多元素标准溶液VI(Merck,目录号:1105800100)
  7. Na 2 N 2 - 乙二胺四乙酸(EDTA)(Carl Roth,目录号:8043.2)
  8. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(Carl Roth,目录号:6763.3)
  9. EDTA洗涤缓冲液(见食谱)


  1. 用于15ml试管的冷却离心机(Beckman Coulter,型号:GS-6)
  2. 铁氟龙杯(Savillex,定制)
  3. 分析天平(KERN& SOHN,型号:ALJ 220-4M)
  4. 涡旋混合器(Eppendorf,型号:MixMate )
  5. 清洁实验室Class 5000(根据美国FED STD 209E,根据更新的ISO 14644-1和ISO 14698标准,将在ISO6和ISO 7之间)
  6. 化学罩(Wesemann)
  7. 热板(陶瓷搅拌热板)(IKA,目录号:35810.01)
  8. ICP-MS 7500cx(安捷伦科技公司,型号:ICP-MS 7500cx)
  9. Neubauer改进的血细胞计数器(Marienfeld-Superior,目录号:0650030)


  1. 取样和清洗(图1)
    1. 将样品放在冰上以减缓代谢活动。
    2. 洗涤步骤是时间敏感的,因为它们通过去除所有外部Mn而停止实验。除去所有的外部锰都保留了样品在所采取的时间点的状态,并且样品的元素组成不再变化。
    3. 在数据分析中计算样品体积和最终洗脱体积。因此,本协议中的所有卷都是大致的数量,只要它们的权重是准确的。所有的转移都可以通过浇注来完成。移液是不必要的,甚至可能会添加污染物。

      A.采样和洗涤的工作流程A.细胞内(样品C)和周质(样品E1和E2)Mn池通过EDTA洗涤步骤分离。 B.为了测量总细胞Mn含量(样品T),将细胞从培养基中分离并重悬于Milli-Q水中。

    1. 对于每个样品,准备四个15毫升的试管,标有样品号和T1(总细胞),E1(第一EDTA洗),E2(第二EDTA洗)和C1(细胞内锰含量)。
    2. 称重并记下每个试管的重量,包括盖子。
    3. 取2ml样品(OD 750〜0.75,注3),并将它们转移到T1和C1管中(注4)。
    4. 从管T1中取出30μl用于细胞计数。
    5. 称重并注意管T1和C1的重量。
    6. 3000×g离心管T1和C1,4℃5分钟。
    7. 丢弃上清。
    8. 通过在约4毫升Milli-Q水中涡旋振荡重悬样品T1。
    9. 向样品C中加入4ml EDTA洗涤缓冲液并通过涡旋重悬。重复步骤1f。
    10. 将EDTA上清液转移到标记为E1的试管中。称重并记下重量。将管E1保存在室温下。样品不需要进一步处理(注5)。
    11. 向样品C中加入4ml EDTA洗涤缓冲液并通过涡旋重悬。重复步骤1f。
    12. 将EDTA上清液转移到标记为E2的试管中。称重并记下重量。将管E2储存在室温下。样本不需要进一步处理。
    13. 加入4毫升Milli-Q水,涡旋重悬。
    14. 从管中取出30μl用于细胞计数。
  2. 样品消化
    1. 对于每个样品,准备另外一套两个15毫升的试管(标有样品编号和C2或T2)。
    2. 称量4毫升的Milli-Q水到试管C2和T2
    3. 每个样品T1和C1加入2毫升硝酸。
    4. 轻轻摇晃或颠倒5分钟。

    5. 在化学罩内预热热板至200°C(注6)
    6. 将每个样品(仅限于T1和C1)放入适合热板的聚四氟乙烯杯中,并将样品置于热板上。
    7. 让样品蒸发,直到最小的液滴保留;
    8. 将相应试管中的所有水加入聚四氟乙烯杯中,小心重新悬浮样品,并将样品返回到同一试管(例如,从管T2到蒸发样品T的水,然后返回管T2)。
  3. 样品测量(不需要洁净室)
    1. 从每个样品(T2,C2,E1和E2)转移2 ml到适合ICP-MS样品处理器的样品管。

    2. 储存每个样品剩余的2毫升,以便进行额外的测量。
    3. 根据您的ICP-MS设备的说明分析样品。
  4. 细胞计数

    1. 根据您的血细胞计数器的使用说明书,对采样和洗涤(步骤1d和1n)样品进行细胞计数。



ICP-MS结果通常以百万分之一(ppm)表示,相当于[μgml -1 -1]或十亿分率(ppb),相当于[μgL -1] 1 ]。确保将输入数据转换为正确的单位。请注意,方程式1和2中的分子量(MW)为[μgmol -1]。
也可以使用OD 750或叶绿素含量来标准化,而不是细胞计数。但是,我们推荐细胞计数。作为对照,样品C2,E1和E2中的Mn的总和应等于样品T2中的Mn浓度。您可以像样本C2一样计算样本T2。

图2.计算Mn浓度的示例确保输入数据的单位是正确的。在我们的例子中,例如,ICP-MS结果是[μgL -1 -1]。请注意,Mn的分子量(MW)是[μgmol -1]。在我们的实验中,E1 + E2 + C2和T2之间的差值在0.1-10×10-3 [原子单元-1]的范围内。


  1. 突变株系基于并与日本的集胞藻 sp进行比较。 PCC 6803,由Martin Hagemann(德国罗斯托克大学)获得。我们的标准生长条件是30℃,100rpm摇动和100μmol光子m-2秒-1 。
  2. 这是一个元素分析,不区分锰结合到蛋白质,复杂的锰和游离锰。
  3. 待分析的原始样品量取决于样品的细胞密度。密集的样品需要更少的体积,稀释的样品需要更多。没有必要调整其他卷。在我们的实验中,我们采取了2毫升的样品,OD 750〜0.75。
  4. 这个协议没有必要使用移液器。这样可以最大限度地减少样品的污染。
  5. 原则上,可以组合样品E1和E2并一起测量它们。如果这样做,则计算中的样本量需要相应地改变。然而,根据实验的生理条件,这些样品中Mn的含量可能已经接近样品E1中的检测极限。因此,我们宁愿不用样品E2进一步稀释样品E1。
  6. 处理大量样品时,热板的温度可以低至180°C。这样做会减慢这个过程,以便更好地跟踪热板上的所有样品,以避免燃烧。
  7. 最小可能的液滴大小主要取决于进行实验的人的经验。重要的是,在测量之前将液滴尺寸包括在样品重量中意味着较大的液滴不会影响测量。这一步骤仅仅是为了通过减少样品中的硝酸量来保护ICP-MS泵。如果样品完全蒸发,加入1毫升硝酸并充分混合。继续小心蒸发。
  8. 所有的样品可以在室温下保存直到消化,只要实验已经停止了洗涤步骤。在消化过程中所有可能的污染都将被消除,但不能改变样品的元素组成。我们没有意识到样品在消化之前有任何特定的保存期限,但是在消化之前我们储存样品的时间最长是三周。消化后,我们建议冷冻样品,以防止可能影响ICP-MS机器的微生物生长,而不是样品成分。


  1. EDTA洗涤缓冲液
    20mM HEPES-KOH,pH 7.5
    5 mM EDTA


这项研究得到了德国科学基金会(EI 945 / 3-1至M.E.)和以色列科学基金会(2733/16至N.K.)的支持。作者声明不存在利益冲突。


  1. Brandenburg,F.,Schoffman,H.,Kurz,S.,Kramer,U.,Keren,N.,Weber,A.P。和Eisenhut,M.(2017)。 Synechocystis 锰出口商Mnx对于蓝藻中的锰稳态是必不可少的。 / a> Plant Physiol 173(3):1798-1810。
  2. Keren,N.,Kidd,M.J。,Penner-Hahn,J.E。和Pakrasi,H.B。(2002)。 在光合细菌集胞藻中大量积累锰的光依赖机制 > sp。 PCC 6803. 生物化学 41(50):15085-15092。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容, 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Brandenburg, F., Schoffman, H., Keren, N. and Eisenhut, M. (2017). Determination of Mn Concentrations in Synechocystis sp. PCC6803 Using ICP-MS. Bio-protocol 7(23): e2623. DOI: 10.21769/BioProtoc.2623.
  2. Brandenburg, F., Schoffman, H., Kurz, S., Kramer, U., Keren, N., Weber, A. P. and Eisenhut, M. (2017). The Synechocystis manganese exporter Mnx is essential for manganese homeostasis in cyanobacteria. Plant Physiol 173(3): 1798-1810.