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2021

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Evaluation of Urine Proteins by Capillary Electrophoresis
毛细管电泳法评价尿蛋白   

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Abstract

Capillary electrophoresis (CE) is a laboratory method usually used to separate proteins in body fluids such as serum, cerebrospinal fluid, or urine. Separation of proteins in urine can have clinical applications for evaluating samples from healthy dogs and dogs with proteinuria in a qualitative way, which would not be possible with gel electrophoresis. Other advantages of CE over gel electrophoresis in serum include the reduced separation time (2 min vs. 20 min in a gel), reduction of waste harmful to humans and the environment, and ability to obtain a curve without the need for additional staining. This protocol is divided into four steps. Firstly, urine needs to be prepared prior to dialysis. Secondly, urine needs to undergo dialysis to eliminate compounds that could interfere with separation, and to concentrate the urine. The third step is CE using specific equipment. The last step is to separate the fractions of the phoretograms obtained in the previous step. This method is mostly an automatized process, easily reproducible, and that can be performed in any laboratory, as a part of the diagnostic or follow-up of patients with renal disease.


Graphical abstract:




Keywords: Canine (犬类), Capillary Electrophoresis (毛细管电泳), Minicap (微型电容), Proteins (蛋白质), Renal (), Sebia (Sebia), Urine (尿液)

Background

Serum protein analysis by capillary electrophoresis (CE) is a well-established laboratory method used for the diagnosis and follow up of infectious, inflammatory, immune-mediated, and neoplastic conditions in human and veterinary medicine (Jenkins, 2009; Giordano and Paltrinieri, 2010). CE is one of the most frequent techniques used to separate serum proteins, as it is a simple, adaptable, and quick technique, and does not require a large sample amount. The CE laboratory technique is based on the separation of charged molecules by their electrophoretic mobility in an alkaline buffer at a specific pH. The separation occurs according to the electrolyte pH and electroosmotic flow, yielding different electrophoretic fractions (Osatinsky, 2007). In serum, the phoretogram is commonly divided into five different fractions, from low to high molecular weight and charge: albumin, alpha1 globulin, alpha2 globulin, beta globulin, and gamma globulin (Gay-Bellile et al., 2003; Tappin et al., 2011).


Recently, the analysis of proteins in urine by CE has proven to be a suitable method in human medicine to detect the presence of characteristic electrophoretic patterns in renal and extrarenal disorders, such as myelomas (Jenkins, 1997; Theodorescu et al., 2005; Mischak et al., 2010). Although quantitative proteinuria can only be assessed by calculation of the urine protein/creatinine ratio (UPC), electrophoretic techniques can be used as a qualitative method to assess the loss of proteins through the urine, as different patterns can be identified (Lees et al., 2005). CE associated with mass spectrophotometry techniques is also useful to identify peptide biomarkers associated with chronic kidney disease (Pelander et al., 2019). The use of urine to identify such abnormalities represents a great advantage over the use of blood, because urine can be collected in quantity, does not require trained staff for its collection, and urinary proteins remain stable for at least 3 days at 4ºC or 3 months at -20°C (Théron et al., 2017). They can provide information about kidney functionality earlier than blood biomarkers, such as SDMA or Cystatin C (Yalçin and Çetin, 2004; Pelander et al., 2019).


Comparison with other electrophoretic techniques that evaluate proteinuria could be challenging because the principle of migration is different. In sodium dodecyl sulfate agarose/polyacrylamide gel (SDS-AGE or SDS-PAGE) electrophoretic techniques, particle migration is only based on their molecular weight. Initially, it might be easier to identify proteins that migrate in the different phoretogram fractions (Yalçin and Çetin, 2004; Zini et al., 2004; Giori et al., 2011; Lavoue et al., 2015; Chacar et al., 2017; Hokamp et al., 2017). Nevertheless, the result of the technique presented in this manuscript is a profile, where abnormalities associated with protein excretions can be easily detected when compared against curves from healthy dogs and any other mammals.


The aim of this study is to establish a standardized protocol to prepare urine adequately, to evaluate the proteins in dog urine by CE on an instrument normally used for serum samples. It can be a useful tool to assess pathological proteinuria in dogs, alongside quantitative methods such as UPC.

Materials and Reagents

  1. Ultrafiltration column 4 mL Vivaspin Turbo 4 10000 MWCO (Sartorius, Vivaspin Turbo 4, catalog number: VS04T02)

  2. Eppendorfs 1.5 mL (Lambda, Eppendorf, catalog number: 1003/G)

  3. Polystyrene tube for urine 12 mL (Lambda, catalog number: 301402)

  4. 10–200 μL tips (Lambda, catalog number: 18260)

  5. Kit Minicap Protein (Sebia Hispania S. A., Sebia, catalog number: 2203). Storage temperature between 2 °C and 30 °C

  6. Kit Urine Dialysis Capillarys (Sebia Hispania S.A., Sebia, catalog number: 2013). Storage temperature between 2 °C and 30 °C

  7. Capiclean (Sebia Hispania S.A., Sebia, catalog number: 2058). Storage temperature between 2 °C and 30 °C

  8. Reconstituted buffer for 4 samples (see Recipes)

Equipment

  1. Minicap + Phoresis system (Sebia Hispania, S.A., catalog number Minicap: 1232; Phoresis. Software version 8.6.3)

  2. Centrifuge Nahita 2650 (Nahita, Nahita 2650, catalog number: 200352650000)

Software

  1. Minicap computer program (Sebia Hispania S.A. www.sebia.com)

Procedure

  1. Preparation of the urine prior to dialysis

    1. Centrifuge 10 mL of urine in a rounded bottom tube at 1,342 × g for 10 min. Identify the tube.

    2. Label the Eppendorf tubes. Transfer and aliquot supernatants obtained in 1.5 mL Eppendorf tubes with a Pasteur pipette. Aliquot a minimum of 4 mL, at 1 mL per Eppendorf. Freeze samples at -20 °C until dialysis.

      Note: As many supernatants as possible should be frozen, in case the dialysis process needs to be repeated. Additionally, 8 mL would be ideal, in case electrophoresis needs to be repeated.


  2. Dialysis and concentration of the samples prior to capillary electrophoresis

    Note: Urine is dialyzed and concentrated using 10 kDa molecular weight cut-off ultrafiltration columns, which are double membranous. This step is necessary not only to concentrate proteins, but also to avoid artifacts from contaminants (Figure 1).



    Figure 1. Different parts of an ultrafiltration colums tube 4 mL capacity.

    The concentration membrane where proteins are retained, and waste deposit where disposals are collected.


    1. Thaw 4 mL of urine supernatant from each individual to study at room temperature, and centrifuge the Eppendorf tubes at 1,609 × g for 10 min.

    2. Transfer this sample to a 4-mL ultrafiltration column. Identify the ultrafiltration columns.

      Note: Columns with 15 mL of capacity are available. In this work, due to the difficulty obtaining a large amount of urine, 4-mL columns are preferred.

    3. Centrifuge the ultrafiltration column with the urine at 1,878 × g for 25 min, or until a maximum volume of 500 μL is left in the column.

    4. Simultaneously, prepare a solution containing 50% distilled water and 50% dialysis buffer (see table of materials) in a sterile container. Use two 10-mL syringes to prepare the solution. Mix this solution prior to its use.

    5. Discard the urine that emerges at the bottom of the column (waste deposit, Figure 1). With a Pasteur pipette, add the washing solution prepared in point 4 to the ultrafiltration column (concentration membrane, Figure 1) with the urine, up to the 4 mL mark.

    6. Centrifuge the column with the urine and the buffer solution at 1,878 × g for 20 min, or until a maximum of 400–500 μL are left in the column.

    7. Homogenize the dialyzed urine with a fine tip micropipette (maximum capacity: 200 mL), and transfer the dialyzed urine to an Eppendorf.

      Note: The capillary electrophoresis equipment (see table of materials) requires a minimum of 100 μL.


  3. Capillary electrophoresis of dialyzed samples

    1. Start the electrophoretic computer program provided by the equipment manufacturer (Phoresis software version 8.6.3). Make sure that the analytical equipment is on too. Enter the program password: a pop-up window will appear and ask to continue with the current technique (protein) or change it to urine. You must change the work mode to urine. Make sure to check reagents and waste deposit before running the samples. A notification will appear on screen when the equipment is ready to process the samples.

      Note: This can be done during the dialysis and concentration of the samples, since the start-up process of the analytical equipment takes 15 min.

    2. Once the equipment is ready, insert the Eppendorf tubes with the dialyzed and concentrated urine into the electrophoresis instrument. Insert samples in pairs to reduce reagent wastage. As soon as the samples are inside, the electrophoretic instrument starts automatically. Do not open the instrument’s door until the CE process is finished.

      Note: The analytical instrument can process 26 samples at the same time, and takes 10 min per sample.


  4. Fraction separation

    1. Phoretograms appear on the screen once the CE process is complete. Identify each phoretogram in the program with the patient’s number. Manually divide the phoretogram into five fractions—although the program establishes predetermined divisions, they are not designed for dog urine (Figure 2).

      Note: The computer program allows manipulations of the profile, such as changing the size or superposition of curves, to be done manually on the phoretograms obtained (Video 1).



    Figure 2. Final result of capillary electrophoresis in urine.

    Phoretogram with the five divisions indicated, from a healthy 2 year entire female Border Collie. Health check was assessed by hematology, biochemistry, serum phoretogram, and complete urinalysis.


    Video 1. Video of how a phoretogram can be divided and how quality control serum (yellow) is superimposed to the study curve (pink).

Recipes

  1. Reconstituted buffer for 4 samples

    Reagent Final concentration Amount
    Dialysis Buffer n/a 8 mL
    Distilled H2O n/a 2 mL
    Total 16 mL

Data analysis

The final result of CE in urine is a profile that represents the different protein fractions contained in dog urine, which varies depending on the amount of protein excreted.

The phoretogram was divided into five fractions based on serum CE. The different fractions that are obtained in each profile are F1—corresponding to albumin, F2—corresponding to alpha1 globulin, F3—corresponding to alpha2 globulin, F4—corresponding to beta globulin, and F5—corresponding to gamma globulin. These fractions were determined by superimposing a normal canine serum sample, diluted 1:49, and used as quality control, over the electrophoretic urine samples (Figure 3). Protein fractions were verified and, if necessary, corrected by visual inspection of the electrophoretogram.



Figure 3. Normal canine diluted serum (yellow) superimposed on the study curve (blue).

The phoretogram is divided into five fractions (F1–F5) according to the diluted canine serum.

Notes

As quality control material, frozen aliquoted serum from a healthy dog was included, diluted 1:49 in running buffer, and migrated prior to any run and in each batch. Internal verification experiments were also performed; within-run and between-run experiments were performed, using urine from a healthy dog. This urine was stored at 4°C during the entire experiment, and dialyzed and concentrated for every run following the manufacturer’s protocol. Three migrations per sample and day (repeatability) were made for five consecutive days (reproducibility), with the objective of calculating the CV for each of the fractions of the urinary proteinogram (Table 1). The CV results were 3.38%, 3.84%, 7.25%, 4.43%, and 7.31% for F1, F2, F3, F4, and F5, respectively. In the between-run experiment, the CV results were 4.78%, 5.17%, 10.0%, 6.09%, and 9.66% for F1, F2, F3, F4, and F5, respectively (Figure 4).


Table 1. Daily coefficient of variation and total coefficient of variation obtained from repeatability (R1, R2, and R3) and reproducibility (Days 1–5) experiments from each fraction (F1–F5).

F1 R1(%) R2(%) R3(%) CVD(%) CVT(%)
Day 1 54 53.2 54 0.86 4.77
Day 2 45.3 49.8 55 9.70
Day 3 54.2 52.1 53.5 2.00
Day 4 53.5 53.4 51.3 2.35
Day 5 54.8 53.2 55.3 2.01
F2 R1(%) R2(%) R3(%) CVD(%) CVT(%)
Día 1 9.6 9.6 9.7 0.60 5.72
Día 2 11.2 11.2 9.6 8.66
Día 3 10.5 9.9 9.4 5.54
Día 4 10.9 10.4 10.1 3.86
Día 5 10.1 10 10.1 0.57
F3 R1(%) R2(%) R3(%) CVD(%) CVT(%)
Día 1 5.8 5.7 5.2 5.77 10
Día 2 6.8 5.7 5.8 9.97
Día 3 5.2 5.6 4.9 6.71
Día 4 5.1 5 5.2 1.96
Día 5 6.6 5.9 5.2 11.86
F4 R1(%) R2(%) R3(%) CVD(%) CVT(%)
Día 1 20.6 21 21 1.10 6.09
Día 2 23.3 22.2 20 7.69
Día 3 20.3 22.2 22.1 4.96
Día 4 19.5 21.2 21.2 4.75
Día 5 18.6 20 19.3 3.62
F5 R1(%) R2(%) R3(%) CVD(%) CVT(%)
Día 1 10 10.5 10.1 2.59 9.66
Día 2 13.4 11.1 9.6 16.83
Día 3 9.8 10.2 10.1 2.07
Día 4 11 10 12.2 9.95
Día 5 9.9 10.9 10.1 5.13

CVD= Daily Coeficient of Variation; CVT= Total Coeficient of Variation; R= Repetition


The limit of detection (LOD) was determined with the urine sample of the same healthy dog. Briefly, 1:2, 1:4, 1:8, and 1:16 dilutions of the dialyzed urine in running buffer were run. The LOD was selected as the last dilution with an electrophoretic pattern equal to the undiluted urine, which was 1:8. The sensitivity obtained in the LOD experiment was 2.1 mg/L, and the initial sample protein concentration was 17.5 mg/L.



Figure 4. Reproducibility and repeatability experiment using a urine from a healthy dog during five consecutive days.

Urine from a healthy dog should be stored at 4 °C, to be dialyzed and concentrated prior to every run.

Acknowledgments

This work was supported by Universidad Católica de Valencia San Vicente Mártir grant (UCV 2016-226-001) (to LG).

This work has been adapted from previous work (Navarro et al., 2021).

Competing interests

The authors have no competing interests to disclose.

Ethics

All experimental procedures have been approved by the research and ethics committee of the Universidad Católica de Valencia San Vicente Mártir (Valencia, Spain; UCV 2017-2018-33).

References

  1. Chacar, F., Kogika, M., Sanches, T. R., Caragelasco, D., Martorelli, C., Rodrigues, C., Capcha, J. M. C., Chew, D. and Andrade, L. (2017). Urinary Tamm-Horsfall protein, albumin, vitamin D-binding protein, and retinol-binding protein as early biomarkers of chronic kidney disease in dogs. Physiol Rep 5(11): e13262.
  2. Gay-Bellile, C., Bengoufa, D., Houze, P., Le Carrer, D., Benlakehal, M., Bousquet, B., Gourmel, B. and Le Bricon, T. (2003). Automated multicapillary electrophoresis for analysis of human serum proteins. Clin Chem 49(11): 1909-1915.
  3. Giordano, A. and Paltrinieri, S. (2010). Interpretation of capillary zone electrophoresis compared with cellulose acetate and agarose gel electrophoresis: reference intervals and diagnostic efficiency in dogs and cats. Vet Clin Pathol 39(4): 464-473.
  4. Giori, L., Tricomi, F. M., Zatelli, A., Roura, X. and Paltrinieri, S. (2011). High-resolution gel electrophoresis and sodium dodecyl sulphate-agarose gel electrophoresis on urine samples for qualitative analysis of proteinuria in dogs. J Vet Diagn Invest 23(4): 682-690.
  5. Hokamp, J. A., Leidy, S. A., Gaynanova, I., Cianciolo, R. E. and Nabity, M. B. (2018). Correlation of electrophoretic urine protein banding patterns with severity of renal damage in dogs with proteinuric chronic kidney disease. Vet Clin Pathol 47(3): 425-434.
  6. Jenkins, M. A. (1997). Clinical application of capillary electrophoresis to unconcentrated human urine proteins. Electrophoresis 18(10): 1842-1846.
  7. Jenkins, M. A. (2009). Serum and urine electrophoresis for detection and identification of monoclonal proteins. Clin Biochem Rev 30(3): 119-122.
  8. Lavoue, R., Trumel, C., Smets, P. M., Braun, J. P., Aresu, L., Daminet, S., Concordet, D., Palanche, F. and Peeters, D. (2015). Characterization of Proteinuria in Dogue de Bordeaux Dogs, a Breed Predisposed to a Familial Glomerulonephropathy: A Retrospective Study. PLoS One 10(7): e0133311.
  9. Lees, G. E., Brown, S. A., Elliott, J., Grauer, G. E., Vaden, S. L. and American College of Veterinary Internal, M. (2005). Assessment and management of proteinuria in dogs and cats: 2004 ACVIM Forum Consensus Statement (small animal). J Vet Intern Med 19(3): 377-385.
  10. Mischak, H., Delles, C., Klein, J. and Schanstra, J. P. (2010). Urinary proteomics based on capillary electrophoresis-coupled mass spectrometry in kidney disease: discovery and validation of biomarkers, and clinical application. Adv Chronic Kidney Dis 17(6): 493-506.
  11. Navarro, P. F., Gil, L., Martin, G. and Fernandez-Barredo, S. (2021). Reference intervals for electrophoretograms obtained by capillary electrophoresis of dialyzed urine from healthy dogs. J Vet Diagn Invest 33(4): 632-639.
  12. Osatinsky, R. (2007). ¿Qué es la electrophoresis capilar? [What is capillary electrophoresis?] Bioquímica y patología clínica. 71: 60-66 Spanish.
  13. Pelander, L., Brunchault, V., Buffin-Meyer, B., Klein, J., Breuil, B., Zurbig, P., Magalhaes, P., Mullen, W., Elliott, J., Syme, H., et al. (2019). Urinary peptidome analyses for the diagnosis of chronic kidney disease in dogs. Vet J 249: 73-79.
  14. Tappin, S. W., Taylor, S. S., Tasker, S., Dodkin, S. J., Papasouliotis, K. and Murphy, K. F. (2011). Serum protein electrophoresis in 147 dogs. Vet Rec 168(17): 456.
  15. Theodorescu, D., Fliser, D., Wittke, S., Mischak, H., Krebs, R., Walden, M., Ross, M., Eltze, E., Bettendorf, O., Wulfing, C., et al. (2005). Pilot study of capillary electrophoresis coupled to mass spectrometry as a tool to define potential prostate cancer biomarkers in urine. Electrophoresis 26(14): 2797-2808.
  16. Théron, M. L., Piane, L., Lucarelli, L., Henrion, R., Layssol-Lamour, C., Palanche, F., Concordet, D., Braun, J. D., Trumel, C. and Lavoue, R. (2017). Effects of storage conditions on results for quantitative and qualitative evaluation of proteins in canine urine. Am J Vet Res 78(8): 990-999.
  17. Yalcin, A. and Çetin, M. (2004). Electrophoretic separation of urine proteins of healthy dogs and dogs with nephropathy and detection of some urine proteins of dogs using immunoblotting. Revue de Medecine Veterinaire 155: 104-112.
  18. Zini, E., Bonfanti, U. and Zatelli, A. (2004). Diagnostic relevance of qualitative proteinuria evaluated by use of sodium dodecyl sulfate-agarose gel electrophoresis and comparison with renal histologic findings in dogs. Am J Vet Res 65(7): 964-971.

简介


[摘要]毛细管电泳(CE)是一种实验室方法,通常用于分离血清、脑脊液或尿液等体液中的蛋白质。尿液中蛋白质的分离可用于以定性方式评估来自健康犬和患有蛋白尿的犬的样本,而这在凝胶电泳中是不可能的。与血清凝胶电泳相比,CE 的其他优势包括缩短分离时间(2 分钟对比凝胶中的 20 分钟)、减少对人类和环境有害的废物,以及无需额外染色即可获得曲线的能力。该协议分为四个步骤。首先,需要在透析前准备尿液。其次,尿液需要进行透析以消除可能干扰分离的化合物,并浓缩尿液。第三步是使用特定设备的 CE。最后一步是分离在上一步中获得的phoretograms的分数。这种方法主要是一个自动化过程,易于重复,可以在任何实验室进行,作为肾病患者诊断或随访的一部分。

图形概要:


[背景] 通过毛细管电泳 (CE) 进行血清蛋白分析是一种成熟的实验室方法,用于诊断和跟踪人类和兽医学中的感染性、炎症性、免疫介导性和肿瘤性疾病(Jenkins,2009;Giordano 和 Paltrinieri,2010 )。 CE 是用于分离血清蛋白的最常用技术之一,因为它是一种简单、适应性强、快速的技术,并且不需要大量样品。 CE 实验室技术基于带电分子在特定pH 值的碱性缓冲液中的电泳迁移率分离。 分离根据电解液的 pH 值和电渗流进行,产生不同的电泳级分 ( Osatinsky , 2007) 。在血清中,色谱图通常分为五个不同的部分,从低到高分子量和电荷:白蛋白、α 1球蛋白、α 2球蛋白、β 球蛋白和 γ 球蛋白(Gay -Bellile 等。 , 2003;塔平 等。 , 2011)。
最近,通过 CE 分析尿液中的蛋白质已被证明是人类医学中检测肾和肾外疾病(例如骨髓瘤)中特征性电泳模式存在的合适方法(Jenkins,1997; Theodorescu 等。 , 2005;米沙克 等。 , 2010)。虽然定量蛋白尿只能通过计算尿蛋白/肌酐比 (UPC) 来评估,但电泳技术可以作为一种定性方法来评估通过尿液丢失的蛋白质,因为可以识别不同的模式 (Lees et al. , 2005)。与质谱技术相关的 CE 也可用于识别与慢性肾病相关的肽生物标志物( Pelander 等。 , 2019)。使用尿液来识别这种异常比使用血液具有很大的优势,因为可以大量收集尿液,不需要经过培训的人员进行收集,并且尿蛋白在 4ºC 或 3 个月内至少保持稳定 3 天或 3 个月在 -20 °C ( Théron 等。 , 2017)。它们可以比血液生物标志物(例如 SDMA 或 Cystatin C)更早地提供有关肾脏功能的信息( Yalçin和Çetin ,2004; Pelander 等。 , 2019)。
与其他评估蛋白尿的电泳技术进行比较可能具有挑战性,因为迁移的原理不同。在十二烷基硫酸钠琼脂糖/聚丙烯酰胺凝胶(SDS-AGE 或 SDS-PAGE)电泳技术中,粒子迁移仅基于其分子量。最初,识别在不同phoretogram分数中迁移的蛋白质可能更容易( Yalçin和Çetin ,2004; Zini 等。 , 2004;焦里 等。 , 2011;拉武 等。 , 2015;查卡尔 等。 , 2017;霍坎普 等。 , 2017)。尽管如此,本手稿中介绍的技术的结果是一个轮廓,与健康狗和任何其他哺乳动物的曲线相比,可以很容易地检测到与蛋白质排泄相关的异常。
本研究的目的是建立一个标准化的方案来充分制备尿液,通过 CE 在通常用于血清样本的仪器上评估狗尿液中的蛋白质。与 UPC 等定量方法一起,它可以成为评估狗的病理性蛋白尿的有用工具。

关键字:犬类, 毛细管电泳, 微型电容, 蛋白质, 肾, Sebia, 尿液



材料和试剂


1. 超滤柱4 mL Vivaspin Turbo 4 10000 MWCO(Sartorius,Vivaspin Turbo 4,目录号: VS04T02)
2. Eppendorfs 1.5 mL(Lambda,Eppendorf,目录号: 1003/克)
3. 用于尿液 12 mL 的聚苯乙烯管(Lambda,目录号: 301402)
4. 10 – 200 μ L吸头(Lambda,目录号: 18260)
5. Kit Minicap Protein( Sebia Hispania SA, Sebia ,目录号:2203)。存储温度在 2 °C和 30 °C之间 
6. 套件尿液透析毛细管( Sebia Hispania SA, Sebia ,目录号:2013)。存储温度在 2 °C和 30 °C之间
7. Capiclean ( Sebia Hispania SA, Sebia ,目录号:2058)。存储温度在 2 °C和 30 °C之间
8. 用于 4 个样品的重组缓冲液(参见配方)


设备


1. Minicap + Phoresis系统( Sebia Hispania, SA,目录号Minicap :1232; Phoresis 。软件版本 8.6.3)
2. 离心机 Nahita 2650( Nahita,Nahita 2650,目录号:200352650000 )


软件


1. Minicap 计算机程序 ( Sebia Hispania SA www.sebia.com )


程序


A. 透析前尿液的制备
1. 在圆形底管中以 1,342 × g离心 10 mL 尿液10 分钟。识别管子。
2. 标记 Eppendorf 管。用巴斯德吸管在 1.5 mL Eppendorf 管中转移和等分上清液。在每个 Eppendorf 1 毫升的情况下,至少分装 4 毫升。将样品冷冻在-20 °C ,直到透析。
冷冻尽可能多的上清液,以防需要重复透析过程。此外,如果需要重复电泳,8 mL 将是理想的。


B. 毛细管电泳前样品的透析和浓缩
注意:使用双膜的 10 kDa 截留分子量超滤柱对尿液进行透析和浓缩。这一步不仅是为了浓缩蛋白质,也是为了避免污染物造成的伪影(图 1)。


 
图 1. 4 mL 容量超滤柱管的不同部分。 
保留蛋白质的浓缩膜和收集废物的废物沉积物。


1. 每个人的 4 mL 尿液上清液,在室温下进行研究,并将 Eppendorf 管以 1,609 × g离心10 分钟。
2. 将此样品转移至4 mL 超滤柱。识别超滤柱。
笔记: 提供容量为 15 mL 的色谱柱。在这项工作中,由于难以获得大量尿液,因此首选 4 mL 色谱柱。
3. 以 1,878 × g将超滤柱与尿液一起离心25 分钟,或直到柱中的最大体积为 500 μL 。
4. 同时,在无菌容器中制备含有 50% 蒸馏水和 50% 透析缓冲液(见材料表)的溶液。使用两个 10 mL 注射器制备溶液。使用前混合此溶液。 
5. 丢弃出现在色谱柱底部的尿液(废物沉积物,图 1)。使用巴斯德吸管,将第 4 点中制备的洗涤溶液与尿液一起添加到超滤柱(浓缩膜,图 1)中,直至 4 mL 标记。
6. 将尿液和缓冲溶液以 1,878 × g离心20 分钟,或直到最多 400 – 500 μL留在柱中。
7. 用细尖微量移液器(最大容量:200 mL )均质化透析尿液,并将透析尿液转移到 Eppendorf中。
注意:毛细管电泳设备(见材料表)至少需要100 μL 。


C. 透析样品的毛细管电泳
1. 启动设备制造商提供的电泳计算机程序 (泳动软件版本 8.6.3)。 确保分析设备也已打开。输入程序密码:会出现一个弹窗询问是否继续 与当前 技术(蛋白质)或将其更改为尿液。您必须将工作模式更改为尿液。确保在运行样品之前检查试剂和废物沉积物。当设备准备好处理时,屏幕上会出现通知 这 样品。  
笔记: 这可以在样品的透析和浓缩过程中完成,因为分析设备的启动过程需要 15 分钟。
2. 一次 这 设备 是 准备好, 插入 这 埃彭多夫 管子 和 这 透析 和 集中 尿 进入 这 电泳 乐器。 插入 样品 在 对 至 减少 试剂 浪费。 立刻 这 样品 是 里面, 这 电泳 乐器 开始 自动地。 做 不是 打开 这 仪器 门 直到 行政长官 过程 是 完成的。
注:分析仪器可同时处理26个样品,每个样品需要10分钟。


D. 馏分分离
1. CE 过程完成后,屏幕上会出现脉象图。 用患者编号识别程序中的每个phoretogram 。手动将phoretogram分为五个部分 - 尽管程序建立了预定的划分,但它们不是为狗尿设计的(图 2)。
phoretograms进行手动操作,例如更改曲线的大小或叠加(视频 1)。


 
图 2。 尿液中毛细管电泳的最终结果。 
泳图,来自健康的2 年整只雌性边境牧羊犬。健康检查通过血液学、生化、血清色谱图和完整的尿液分析进行评估。


 
视频 1. 如何划分phoretogram以及如何将质量控制血清(黄色)叠加到研究曲线(粉红色)上的视频。


食谱


1. 用于 4 个样品的重组缓冲液
试剂 最终浓度 数量
透析缓冲液 不适用 8 毫升
蒸馏 H 2 O 不适用 2 毫升
全部的 16 毫升


数据分析


尿液中 CE的最终结果是代表狗尿中所含的不同蛋白质部分的概貌,这取决于排泄的蛋白质量。
将phoretogram分为五个部分。在每个配置文件中获得的不同部分是 F1 -对应于白蛋白,F2 -对应于 alpha 1球蛋白,F3 -对应于 alpha 2球蛋白,F4 -对应于 β 球蛋白,F5 -对应于丙种球蛋白。这些分数是通过将正常的犬血清样品(按 1:49 稀释并用作质量控制)叠加在电泳尿液样品上来确定的(图 3)。验证蛋白质级分,如有必要,通过电泳图的目视检查进行校正。  


 
图 3. 叠加在研究曲线(蓝色)上的正常犬稀释血清(黄色)。
根据稀释的犬血清,色谱图分为五个部分(F1-F5)。


笔记


作为质量控制材料,包括来自健康狗的冷冻等分血清,稀释 1:49 在运行缓冲区中,并在任何运行之前和每批中迁移。还进行了内部验证实验;使用来自健康狗的尿液进行运行内和运行间实验。在整个实验过程中,将尿液储存在 4 °C ,并按照制造商的方案在每次运行时进行透析和浓缩。连续五天(可重复性)对每个样品和每天进行 3 次迁移(可重复性),目的是计算尿蛋白图每个部分的 CV(表 1)。 F1、F2、F3、F4 和 F5 的 CV 结果分别为 3.38%、3.84%、7.25%、4.43% 和 7.31%。在运行间实验中,F1、F2、F3、F4 和 F5 的 CV 结果分别为 4.78%、5.17%、10.0%、6.09% 和 9.66%(图 4)。


表 1. 从每个馏分 (F1-F5) 的重复性(R1、R2 和 R3)和再现性(第 1-5 天)实验中获得的每日变异系数和总变异系数。
F1 R1(%) R2(%) R3(%) CV D (%) CV T (%)
第 1 天 54 53.2 54 0.86 4.77
第 2 天 45.3 49.8 55 9.70
第 3 天 54.2 52.1 53.5 2.00
第 4 天 53.5 53.4 51.3 2.35
第 5 天 54.8 53.2 55.3 2.01
F2 R1(%) R2(%) R3(%) CV D (%) CV T (%)
迪亚 1 9.6 9.6 9.7 0.60 5.72
迪亚 2 11.2 11.2 9.6 8.66
迪亚 3 10.5 9.9 9.4 5.54
迪亚 4 10.9 10.4 10.1 3.86
迪亚 5 10.1 10 10.1 0.57
F3 R1(%) R2(%) R3(%) CV D (%) CV T (%)
迪亚 1 5.8 5.7 5.2 5.77 10
迪亚 2 6.8 5.7 5.8 9.97
迪亚 3 5.2 5.6 4.9 6.71
迪亚 4 5.1 5 5.2 1.96
迪亚 5 6.6 5.9 5.2 11.86
F4 R1(%) R2(%) R3(%) CV D (%) CV T (%)
迪亚 1 20.6 21 21 1.10 6.09
迪亚 2 23.3 22.2 20 7.69
迪亚 3 20.3 22.2 22.1 4.96
迪亚 4 19.5 21.2 21.2 4.75
迪亚 5 18.6 20 19.3 3.62
F5 R1(%) R2(%) R3(%) CV D (%) CV T (%)
迪亚 1 10 10.5 10.1 2.59 9.66
迪亚 2 13.4 11.1 9.6 16.83
迪亚 3 9.8 10.2 10.1 2.07
迪亚 4 11 10 12.2 9.95
迪亚 5 9.9 10.9 10.1 5.13
CV D = 每日变异系数; CV T = 总变异系数; R= 重复


检测限 (LOD) 是用同一只健康狗的尿液样本确定的。简而言之,在运行缓冲液中运行 1:2、1:4、1:8 和 1:16 稀释的透析尿液。选择 LOD 作为最后一次稀释,其电泳模式等于未稀释的尿液,即 1:8。 LOD实验得到的灵敏度为2.1 mg/L,初始样品蛋白浓度为17.5 mg/L。


 
连续五天使用健康狗的尿液进行的可重复性和可重复性实验。
健康狗的尿液应储存在 4 °C,每次跑步前进行透析和浓缩。


致谢


这项工作得到了瓦伦西亚天主教大学San Vicente Mártir赠款 (UCV 2016-226-001) (给 LG) 的支持。
这项工作改编自以前的工作(Navarro等人,2021 年)。


竞争利益


作者没有竞争利益要披露。


伦理


所有实验程序均已获得瓦伦西亚天主教大学圣维森特马尔蒂尔(西班牙瓦伦西亚;UCV 2017-2018-33)研究和伦理委员会的批准。


参考


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引用:Navarro, P. F., Gil, L. and Férnandez-Barredo, S. (2022). Evaluation of Urine Proteins by Capillary Electrophoresis. Bio-protocol 12(15): e4466. DOI: 10.21769/BioProtoc.4466.
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