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Fish Bile Clean-up for Subsequent Zymography and Mass Spectrometry Proteomic Analyses
鱼胆汁清除后进行酶谱和质谱蛋白质组学分析   

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Aquatic Toxicology
Apr 2012

Abstract

Biliary excretion offers a way to analyze various contaminants in aquatic organisms, and fish bile has been used as a biomarker for environmental contamination. The use of the fish bile proteome as a tool for monitoring the impact of environmental contaminants has been recently validated. However, scarce studies in this context are available, and much remains to be investigated. In this context, this protocol describes a fast, reproducible and cheap biliary clean-up procedure for subsequent proteomic analyses, such as zymography and mass spectrometry.

Keywords: Fish bile (鱼胆汁), Purification (纯化), Delipidation (脱脂), Desalting (脱盐), Proteomics (蛋白质组学), Zymography (酶谱), Mass spectrometry (质谱)

Background

Biliary excretion is an alternative way to analyze several chemical pollutants in aquatic organisms. Fish bile has been routinely applied as a biomarker for environmental contamination for several decades, although the focus until recently has been only the detection of environmental contaminants, and not the evaluation of possible biochemical effects, such as differential protein expression. However, the fish bile proteome has been recently validated as a tool for monitoring the impact of environmental contaminants on fish metabolism. These include the possible deleterious effects of polycyclic aromatic hydrocarbons (Pampanin et al., 2014), metals and metalloids (Hauser-Davis et al., 2012a) and complex mixtures (Hauser-Davis et al., 2012b), among other pollutants. However, this is a very recent field, and studies are still lacking in this regard. In this context, fish bile contains high amounts of lipids and bile salts, which interfere in protein quantification, 1D- and 2D-electrophoresis and mass spectrometry analyses (Hauser-Davis et al., 2012b). Thus, clean-up procedures are of the utmost importance in order to obtain reproducible results and adequately prepare this fluid for subsequent proteomic applications.

Materials and Reagents

  1. Pipette tips (Corning, Axygen®, catalog number: T-1005-WB-C )
  2. 2 ml polypropylene microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-200-C )
  3. Sterile syringes (3 ml or higher)
  4. Vivaspin 500 concentrators, MWCO 3 kDa (Sartorius, catalog number: VS0191 )
  5. Fish
  6. Alcohol (100%)
  7. Cleanascite® (Biotech Support Group, catalog number: X2555-50 )

Equipment

  1. Stainless steel scissors
  2. Pipette 100-1,000 µl (Eppendorf, catalog number: 3120000267 )
  3. Ultrasonic water bath (DK SONIC, model: DK-80 )
  4. Refrigerated centrifuge (Eppendorf, model: 5415 R )
  5. Orbital shaker (Wincom/OEM/Neutral, model: TS-2000A )

Procedure

A flowchart describing the biliary clean-up procedure is displayed below in Figure 1.


Figure 1. Flowchart describing the biliary clean-up procedure

  1. Fish dissection procedure
    1. Before each dissection, the stainless steel scissors should be cleaned with alcohol (100%) to avoid contamination.
    2. An incision is made from the urogenital pore up to the gills using a stainless steel scissors. After the incision is made, the operator, using both hands, may split open the fish to expose the internal organs, more specifically, the liver.
    3. After locating the liver, the operator should move his/her fingers behind this organ, in order to locate the gallbladder, without causing any mechanical damage to this organ (Figure 2). It is important to note that some fish species either do not possess a gallbladder, or it is too small to collect an adequate bile volume.


      Figure 2. Fish gallbladder, pointed out by the scissors tip, usually located behind the liver. The gallbladder in question is full, with light green bile.

    4. After locating the gallbladder, sterile syringes are used to puncture the gallbladder and obtain the biliary fluid. Remove as much of the fluid as possible.
    5. Bile color and volume should be recorded, and the samples should immediately be aliquoted into 500 μl aliquots in sterile 2 ml polypropylene microcentrifuge tubes and frozen if not processed at once (preferably, at -80 °C. If an ultra-freezer is not available, samples may be frozen at -20 °C).

  2. Bile processing
    1. Bile samples are sonicated for 5 min in order to break down cellular membranes. The use of a cold water-bath is preferred. If one is not available, the microcentrifuge tube should be removed every 20-30 sec from the bath, to avoid over-heating.
    2. After sonication, the samples are centrifuged for 15 min at 16,000 x g (4 °C), in order to remove cellular debris. There is no need to pre-cool the microcentrifuge tubes, since centrifugation is conducted at 4 °C and the next step is conducted on ice.
    3. After the centrifugation, transfer the sample supernatants to new sterile 2 ml polypropylene microcentrifuge tubes. Probably no pellet will be visible, only a slight slick film stuck to the tube wall (Step B2). Separate the supernatant anyway.
    4. The samples are then delipidized to avoid interferences in downstream proteomic applications.
      The commercial solid-phase, non-ionic adsorbent lipid removal agent, Cleanscite® is used for this purpose, according to manufacturer’s instructions. This consists of adding a total of 75 μl of this reagent to 300 μl of the sample. (These volumes may be reduced, but the Cleanscite® solution:sample ratio should be maintained). It is important to shake the Cleanscite® solution prior to application, since the detergent may have slightly solidified.
    5. After adding the Cleanscite®, samples are shaken on an orbital shaker, on ice, for 1 h and then centrifuged for 1 min at 16,000 x g (4 °C) to precipitate the lipids.
    6. After centrifugation, transfer the sample supernatants to new sterile polypropylene microcentrifuge tubes. The pellet will, again, probably only be a slight slick film stuck to the tube wall (Step B5). Separate the supernatant anyway.
    7. The samples are then desalted, since salts interfere with downstream proteomic applications, and concentrated, by using Vivaspin® (or similar) concentrators. The molecular weight cut-off used depends on the aim of the study, in this case, 3 kDa. When using Vivaspin 500®, add a maximum volume of 500 μl of the sample to the concentrator, and centrifuge at 15,000 x g for 30 min (4 °C). Subsequently, transfer the liquid remaining at the top of the concentrator to new sterile polypropylene microcentrifuge tubes.
    8. Samples are ready for downstream proteomics applications, such as zymography and mass spectrometry.

Data analysis

The adequacy of the sample clean-up procedure can be verified by the following:

  1. Determine the total protein content by Bradford, Lowry or any other usual method. Before sample clean-up, inconsistent data is obtained for total protein content in bile, due to the presence of high amounts of lipids and bile salts, with extremely high replicate variability. After clean-up, replicates should be reproducible.
  2. Run a 1D protein SDS-PAGE electrophoresis or zymogram gel to check for smearing (see Hauser-Davis et al. [2012b], Figure 3, for an example visualized on a 10 % gelatin zymogram of an inadequate sample, displaying extreme smearing and few apparent lytic bands, and Figures 4 and 5 for adequate results).

Notes

  1. Fish should be obtained as fresh as possible, to avoid proteolytic degradation of the bile samples.
  2. Bile volume and color should be recorded if inferences regarding food status and exposure time are desired (Hauser-Davis, 2017).
  3. A avoid repeated freeze-thaw cycles of the samples (not more than 5 times), as usual in proteomic applications, in order to reduce irreproducibility.

Acknowledgments

This protocol is adapted from Hauser-Davis et al. (2012b). There are no conflicts of interest or competing interests. The author would like to thank the Brazilian National Council for Scientific and Technological Development (CNPq) for financial support.

References

  1. Hauser-Davis, R. A. (2017). Characterization of biliary caseinolytic proteases in two environmental contamination sentinel fish species with an emphasis on metalloproteinases. In: Daniels, J. A. (Ed.). Advances in Environmental Research. Volume 55 pp: 101-126.
  2. Hauser-Davis, R. A., Goncalves, R. A., Ziolli, R. L. and de Campos, R. C. (2012a). A novel report of metallothioneins in fish bile: SDS-PAGE analysis, spectrophotometry quantification and metal speciation characterization by liquid chromatography coupled to ICP-MS. Aquat Toxicol 116-117: 54-60.
  3. Hauser-Davis, R. A., Lima, A. A., Ziolli, R. L. and Campos, R. C. (2012b). First-time report of metalloproteinases in fish bile and their potential as bioindicators regarding environmental contamination. Aquat Toxicol 110-111: 99-106.
  4. Pampanin D. M., Larssen E., Øysæd K. B., Sundt R. C. and Sydnes, M. O. (2014). Study of the bile proteome of Atlantic cod (Gadus morhua): Multi-biological markers of exposure to polycyclic aromatic hydrocarbons. Mar Environ Res 101: 161-168.

简介

胆汁排泄提供了分析水生生物中各种污染物的方法,并且鱼胆已被用作环境污染的生物标志物。 最近验证了使用鱼胆蛋白质组作为监测环境污染物影响的工具。 然而,在这方面缺乏研究,还有很多需要调查。 在这种情况下,这个协议描述了一个快速,可重复和廉价的胆道清理程序后续蛋白质组学分析,如酶谱和质谱。

【背景】胆汁排泄是分析水生生物中几种化学污染物的一种替代方法。虽然直到最近,焦点只是检测环境污染物,而不是评估可能的生物化学效应,例如差异蛋白质表达,鱼胆汁已被常规用作环境污染的生物标志物数十年。然而,鱼胆汁蛋白质组最近被验证作为监测环境污染物对鱼代谢的影响的工具。这些包括多环芳香烃(Pampanin等人,2014),金属和类金属(Hauser-Davis等人,2012a)和复杂混合物( Hauser-Davis et al。,2012b)等污染物。但是,这是一个很新的领域,在这方面还缺乏研究。在这种情况下,鱼胆含有大量的干扰蛋白定量,1D和2D电泳和质谱分析(Hauser-Davis等人,2012b)的脂质和胆汁盐。因此,为了获得可重现的结果,清理过程是至关重要的,并为随后的蛋白质组学应用充分准备这种液体。

关键字:鱼胆汁, 纯化, 脱脂, 脱盐, 蛋白质组学, 酶谱, 质谱

材料和试剂

  1. 移液器吸头(Corning,Axygen <\ sup>,产品目录号:T-1005-WB-C)
  2. 2毫升聚丙烯微量离心管(Corning,Axygen,目录号:MCT-200-C)
  3. 无菌注射器(3毫升或更高)

  4. Vivaspin 500浓缩器,MWCO 3 kDa(Sartorius,目录号:VS0191)

  5. 酒精(100%)
  6. (生物技术支持组,目录号:X2555-50)

设备

  1. 不锈钢剪刀
  2. 移取100-1,000μl(Eppendorf,目录号:3120000267)
  3. 超声波水浴(DK SONIC,型号:DK-80)
  4. 冷冻离心机(Eppendorf,型号:5415 R)
  5. 轨道振动器(Wincom / OEM / Neutral,型号:TS-2000A)

程序

描述胆道清理程序的流程图如图1所示。


图1.描述胆道清理程序的流程图

  1. 鱼解剖程序
    1. 在每次解剖之前,不锈钢剪刀应该用酒精(100%)清洁以避免污染。
    2. 使用不锈钢剪刀从泌尿生殖器毛孔切至鳃。在切开之后,操作者用双手可以将鱼分开,以暴露内脏器官,更具体地是肝脏。
    3. 在找到肝脏之后,操作者应该将手指移到该器官的后面,以便定位胆囊,而不会对该器官造成任何机械损伤(图2)。重要的是要注意,有些鱼类或者不拥有胆囊,或者胆囊太小而无法获得足够的胆汁量。


      图2.由剪刀尖指出的鱼胆,通常位于肝脏后面。胆囊充满胆汁,胆汁呈浅绿色。

    4. 定位胆囊后,使用无菌注射器刺穿胆囊并获得胆汁。尽可能多地去除流体。
    5. 应记录胆汁的颜色和体积,立即将样品分装到无菌2毫升聚丙烯微量离心管中,分装成500微升等分试样,如果不立即处理,最好冷冻(最好是在-80℃,如果没有超冷冻器,样品可能在-20°C冷冻)。

  2. 胆汁加工
    1. 将胆汁样品超声处理5分钟以分解细胞膜。使用冷水浴是优选的。如果没有可用的话,应该每隔20-30秒从浴中移出微量离心管,以避免过热。
    2. 超声处理后,将样品在16,000×g(4℃)下离心15分钟以除去细胞碎片。不需要预冷微量离心管,因为在4℃下进行离心分离,下一步在冰上进行。
    3. 离心后,将样品上清液转移到新的无菌2ml聚丙烯微量离心管中。可能没有颗粒可见,只有轻微的光滑的薄膜粘在管壁上(步骤B2)。无论如何分离上清液。
    4. 然后将样品脱脂以避免在下游蛋白质组学应用中的干扰。
      根据制造商的说明,使用商业固相非离子吸附脂质去除剂Cleanscite。这包括将总共75μl的该试剂加入到300μl的样品中。 (这些体积可能会减少,但Cleanscite®解决方案:样品比应保持不变)。在使用前摇动Cleanscite 溶液非常重要,因为清洁剂可能稍微固化。
    5. 加入Cleanscite后,将样品在定轨摇床上摇动1小时,然后在16,000×g(4℃)下离心1分钟以沉淀脂质。
    6. 离心后,将样品上清液转移到新的无菌聚丙烯微量离心管中。颗粒将, 再次,可能仅仅是粘在管壁上的轻微的光滑膜(步骤B5)。无论如何分离上清液。
    7. 然后将样品脱盐,因为盐会干扰下游的蛋白质组学应用,并通过使用Vivaspin(或类似的)浓缩器浓缩。使用的分子量截止取决于研究的目的,在这种情况下,3kDa。当使用Vivaspin 500™时,向样品浓缩器中加入500μl样品的最大体积,并在15,000×g的条件下离心30分钟(4℃)。随后,将剩余在浓缩器顶部的液体转移到新的无菌聚丙烯微量离心管中。
    8. 样品已准备好用于下游蛋白质组学应用,如酶谱法和质谱法。

数据分析

样品清理程序的适当性可以通过以下方法来验证:

  1. 通过Bradford,Lowry或任何其他常规方法确定总蛋白质含量。在样品净化之前,由于存在大量的脂质和胆汁盐,因此获得胆汁中总蛋白质含量的不一致的数据,具有极高的重复变异性。清理后,重复应该是可重复的。
  2. 运行1D蛋白质SDS-PAGE电泳或酶谱凝胶以检查涂片(参见Hauser-Davis等人[2012b],图3,对于在10%明胶酶谱图上显示的不足样品显示出极端的涂片和少量明显的裂解条带,图4和图5为合适的结果)。

笔记

  1. 应尽可能新鲜地获得鱼,以避免胆汁样品的蛋白水解降解。
  2. 如果需要对食物状况和暴露时间进行推断,则应记录胆汁的体积和颜色(Hauser-Davis,2017)。
  3. 避免反复冻融样品(不超过5次),如通常的蛋白质组学应用,以减少不可再现性。

致谢

这个协议是从豪瑟 - 戴维斯等人改编的。 (2012B)。没有利益冲突或利益冲突。作者要感谢巴西国家科学和技术发展委员会(CNPq)的财政支持。

参考

  1. Hauser-Davis,R.A。(2017)。 在两种环境污染哨点鱼种中鉴定胆汁酪蛋白水解蛋白酶,重点是金属蛋白酶。 / a>在:Daniels,JA(Ed。)。环境研究进展。第55卷:101-126。
  2. Hauser-Davis,R.A.,Goncalves,R.A。,Ziolli,R.L。和de Campos,R.C。(2012a)。 鱼胆中金属硫蛋白的新颖报告:SDS-PAGE分析,分光光度法定量和金属形态表征液相色谱耦合到ICP-MS。 Aquat Toxicol 116-117:54-60。
  3. Hauser-Davis,R.A.,Lima,A.A。,Ziolli,R.L。和Campos,R.C。(2012b)。 首次报道鱼胆汁中的金属蛋白酶及其作为环境污染的生物指标的潜力 Aquat Toxicol 110-111:99-106。
  4. Pampanin D. M.,Larssen E.,ØysædK. B.,Sundt R.C和Sydnes,M.O.(2014)。 研究大西洋鳕鱼胆汁蛋白质组( Gadus morhua ):多暴露于多环芳烃的生物标志物。


    Mar Environ Res 101:161-168。

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hauser-Davis, R. (2018). Fish Bile Clean-up for Subsequent Zymography and Mass Spectrometry Proteomic Analyses. Bio-protocol 8(2): e2706. DOI: 10.21769/BioProtoc.2706.
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