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Mass Spectrometry-based in vitro Assay to Identify Drugs that Influence Cystine Solubility
基于质谱法的体外实验鉴定影响胱氨酸溶解度的药物   

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Abstract

Cystinuria is a rare genetic disorder characterized by recurrent, painful kidney stones, primarily composed of cystine, the dimer of the amino acid cysteine (Sumorok and Goldfarb, 2013). Using a mouse model of cystinuria, we have recently shown that administration of drugs that increase cystine solubility in the urine can be a novel therapeutic strategy for the clinical management of the disease (Zee et al., 2017). There is a large unmet need in the field for developing new drugs for cystinuria. To that end, here we describe a simple in vitro cystine solubility assay that is amenable for screening compounds to identify potential drugs that may influence cystine solubility. The assay includes preparing a supersaturated solution of cystine, incubating this solution with drug(s) of choice, and finally using high pressure liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) to quantify the amount of cystine precipitated under various conditions.

Keywords: Cystinuria(胱氨酸尿), Kidney stones(肾结石), Cystine(胱氨酸), Mass spectrometry(质谱法), Drug screening(药物筛查), L-cystine dimethyl ester(L-胱氨酸二甲酯)

Background

Cystinuria is a type of kidney stone disease characterized by a genetic defect in cystine transporters in the proximal tubule of the kidney, resulting in massive increase in cystine load in the urine that precipitate as kidney stones (Sumorok and Goldfarb, 2013). While categorized as a rare genetic disorder (~1/15,000 individuals) (Milliner and Murphy, 1993; Palacin et al., 2001), patients suffering from cystinuria experience excruciating pain from recurrent stone episodes (Dent and Senior, 1955). Unlike other, more common kidney stone types (such as calcium oxalate- or uric acid-based), cystine stones are denser and resistant to extracorporeal shock wave lithotripsy (SWL), requiring patients to undergo multiple emergency room visits and surgical procedures to remove obstructive stones (Mattoo and Goldfarb, 2008). The current drug regimen for cystinuria are geared towards either increasing urinary pH (potassium citrate) or reducing urinary cystine levels (thiol drugs, such as tiopronin), which are generally associated with serious side effects (Koraishy et al., 2013; Sumorok and Goldfarb, 2013; Saravakos et al., 2014). Further, clinical (Becker et al., 2007) and murine model (Zee et al., 2017) studies have found little evidence that these drugs are ultimately effective and long-term patient compliance is poor. Thus, there is an urgent need for developing effective therapies to treat cystinuria. Our recent results show that increasing urinary cystine solubility can be a viable alternative strategy for drug development in cystinuria. Herein, we describe an in vitro cystine solubility assay to identify novel compounds capable of influencing cystine solubility.

Materials and Reagents

  1. Gloves
  2. Lab coat
  3. Culture tubes (16 x 100 mm) (VWR, catalog number: 47729-576 )
  4. 4 ml glass vials and caps (13-425 thread finish, borosilicate glass) (VWR, catalog number: 46610-706 )
  5. HPLC autosampler vials and caps (9-425 thread finish) (VWR, catalog number: 89523-482 )
  6. Pipette tips (standard 1,000 µl and 200 µl)
  7. Gel loading pipette tips (VWR, catalog number: 37001-150 )
  8. Joint clips to attach the round bottom flask to Allihn condenser (VWR, catalog number: 89426-304)
    Manufacturer: GLASSCOLABS, catalog number: 007.470.05A .
  9. Eppendorf tubes (standard 1.5 ml)
  10. Ammonium acetate (for LC-MS LiChropur®) (EMD Millipore, catalog number: 533004 )
  11. Water (HPLC-grade) (VWR, BDH®, catalog number: BDH23595.400 )
  12. Ammonium hydroxide (28% NH3 in H2O, ≥ 99.99%) (Sigma-Aldrich, catalog number: 338818 )
  13. 3,3,3’,3’-d4-L-cystine (98%) (Cambridge Isotope Laboratories, catalog number: DLM-1000-1 )
  14. L-cystine (≥ 98%) (Sigma-Aldrich, catalog number: C8755 )
  15. DL-cystine dimethyl ester dihydrochloride (≥ 95%) (Sigma-Aldrich, catalog number: 857327 )
  16. Acetonitrile (HPLC-grade) (VWR, BDH®, catalog number: BDH83639.400 )
  17. Ammonium acetate solution (see Recipes)

    Note: Specific brand and catalog number (as used in our work) have been provided for the materials and reagents listed. However, we do not explicitly endorse any of these brands or products and most items can be obtained from other reputed vendors. Further, most of these product/catalog numbers are based on the current availability in the United States, so researchers in other countries may find it necessary to obtain materials from another vendor.

Equipment

  1. pH meter (standard)
  2. Safety goggles
  3. Chemical safety hood
  4. Glass bottles (standard 500 ml and 1 L)
  5. Glass measuring cylinders (standard 1 L, 250 ml, and 100 ml)
  6. Glass round bottom flask (standard 24/40 ground joint, 100 ml)
  7. Magnetic stir-bar (standard)
  8. Reflux apparatus (standard, consisting of stirring-heating mantle, Allihn condenser with a water jacket and 24/40 junction, running water source, rubber tubing, and clips for attachment). Follow vendor instructions for assembly
  9. Pipettes (standard 1,000 µl and 200 µl)
  10. Vortex (standard)
  11. -20 °C freezer (standard)
  12. 4 °C incubator or fridge (standard)
  13. HPLC-MS system:
    1. HPLC: Shimadzu UFLC prominence system (HPLC) (Shimadzu, model: Prominence UFLC ) fitted with following modules: CBM-20A (Communication bus module), DGU-A3 (degasser), two LC-20AD (liquid chromatograph, binary pump), SIL-20AC HT (auto sampler)
    2. HPLC column: Luna® NH2 column (2 x 50 mm, 3 µm, 100 Å) (Phenomenex, catalog number: 00B-4377-B0 )
    3. Mass Spectrometer: AB Sciex 4000 LC-MS/MS mass spectrometer (AB Sciex, model: QTRAP® 4000 ) fitted with a Turbo VTM ion source
  14. Centrifuge (standard, with attachment for 4 ml vials)
  15. Weighing scale (standard)

    Note: Standard lab equipment should be used for this assay as indicated. Specific brand and catalog numbers (as used in our work) have been provided for components of the HPLC-MS setup–these can be replaced by equipment from other vendors; arguably any HPLC-MS setup will work with this assay, but may need additional optimization based on vendor-provided instructions.

Software

  1. AB Sciex’s Analyst® v1.6.1 for data acquisition, development of LC method, and optimization of analyte-specific MRM (multiple reaction monitoring) transitions
  2. AB Sciex’s Peakview® v2.1 and Skyline® v3.623 for LC-MS/MS data analysis

Procedure

  1. Preparation of solutions and drug standards
    1. Prepare 2 L of 20 mM ammonium acetate in HPLC-grade water. This will be used for HPLC sample preparation, as well as to make one of the HPLC mobile phases.
      Note: 2 L is a suggested volume, adjust based on the anticipated number of drugs being tested.
    2. Adjust pH of this solution to ~9.0-9.5 (this does not have to be accurate or reproducible) by adding ammonium hydroxide in batches of ~0.5 ml followed by pH measurement. A standard pH meter can be used for the purpose; however, it is recommended that the pH probe not be dipped directly in the solution. Instead, a small aliquot (~0.5-1 ml) should be transferred to a culture tube for each round of pH measurement.
      Caution: Ammonium hydroxide is highly corrosive; gloves, safety goggles, and lab coat should be worn at all times while handling this compound. It is also advisable to handle it only inside a chemical safety hood.
    3. Measure 950 ml of this pH-adjusted 20 mM ammonium acetate solution in a clean glass bottle (1 L) and add 50 ml of acetonitrile (ACN), using appropriate glass cylinders. This will be used as mobile phase A for HPLC.
      Note: This solution can be stored for ~2 weeks at room temperature. Bottles and glass cylinders should be thoroughly cleaned first with HPLC water and then with ACN prior to their use (detergents/soaps should be strictly avoided).
    4. Measure 1 L of ACN to another clean glass bottle (1 L). This will be used as mobile phase B for HPLC.
      Note: ACN can be stored in the dark at room temperature and used for up to 6 months.
    5. Take 400 ml pH-adjusted 20 mM ammonium acetate solution (from step A2) in a 500 ml glass bottle. Weight out ~29.2 mg of 3,3,3’,3’-d4-L-cystine (referred to as d4-CC) into this solution and mix well to dissolve and generate a 300 µM solution of d4-CC.
      Note: This solution can be aliquoted and stored for several months at -20 °C.
    6. Prepare adequate amount of 30 µM d4-CC solution from this 300 µM solution of d4-CC, by diluting (10 folds) with pH-adjusted 20 mM ammonium acetate solution (from step A2).
      Note: This solution should be prepared fresh for each batch. 3.6 ml of this solution will be used as internal standard per vial used in the assay–calculate the amount required based on the number of drugs (and replicates) being tested.
    7. Prepare 500 µl each of 2.5 mM and 250 µM solutions of DL-cystine dimethyl ester (CDME) in HPLC-grade water (5x). CDME is used as a positive control for the assay as it has been shown to significantly increase cystine solubility (Rimer et al., 2010; Zee et al., 2017).
    8. Prepare test drugs solutions (5x): weigh out test drugs and dissolve in HPLC-grade water to achieve concentrations 5 folds higher than intended for testing; in the course of the assay, this will be diluted to 1x.
      Caution: The drugs may pose health and/or environmental hazards; thus, they should be handled with proper safety precautions.
    9. Prepare the positive control vials (4 replicates each): two sets of 4 x 4 ml glass vials with 100 µl each of 2.5 mM and 250 µM solutions of CDME (from step A7). In the course of the assay, this will be diluted 5 folds to achieve a final concentration of 500 µM and 50 µM CDME respectively.
    10. Prepare the negative control vials (4 replicates each): one set of 4 x 4 ml glass vials with 100 µl each of HPLC water.
    11. Prepare the drug vials (4 replicates each): one set of 4 x 4 ml glass vials with 100 µl each of 5x drug solution (from step A8). In the course of the assay, this will be diluted to 1x.

      Notes:

      1. For steps A9-A11, at least 3 replicates (ideally 4 replicates) per condition should be used.
      2. Borosilicate glass vials as indicated are absolutely required for this step; plastic vials are not compatible with this assay.

  2. Preparation of supersaturated solution of cystine
    1. Weight out ~96 mg of L-cystine (referred to as CC) in a round bottom flask and add 100 ml of HPLC-grade water to achieve a final concentration of 4 mM of CC.
      Notes:
      1. Wash round bottom flask thoroughly with HPLC-grade water, ACN, and water again. Avoid the use of soaps/detergent.
      2. CC will not dissolve at this stage.
      3. 100 ml is a suggested volume, adjust based on anticipated number of drugs being tested. Here is an exemplary scenario when 10 drugs are being tested at two concentrations (4 replicates per condition): in this case, one will have 80 drug vials (10 drugs x 2 concentrations x 4 replicates each) and an additional 12 vials for the positive and negative controls to be used in each setup (see steps A9 and A10), i.e., a total of 92 x 4 ml vials. For such a setup, one would need a total of 92 x 400 µl, i.e., 36.8 ml of 4 mM CC solution (vide infra, step C1). Thus preparing 50 ml of solution for reflux (by weighing ~48 mg CC) would suffice.
    2. To this add a magnetic stir bar (of suitable size).
    3. Attach the round bottom flask containing the stir bar and CC suspension to a reflux apparatus. Securely attach the flask to the Allihn condenser using clips and turn on the water flow through the condenser jacket. Ensure that a steady (but not vigorous) flow of water is achieved through the condenser.
      Note: Refluxing refers to the process of heating a solvent (or suspension) to its boiling point and allowing the evaporated solvent to condense back into the original flask, most often using cold water flowing through an Allihn condenser. Refluxing ensures that the solvent (or suspension) is constantly maintained at its boiling point (a fixed temperature), for a prolonged period, without any significant solvent loss due to evaporation.
      Caution: The reflux apparatus should be set up inside a chemical safety hood. The process requires heating at high temperatures, thus appropriate safety precautions should be taken.
    4. When the water stream through the reflux condenser is steady, turn on the stirring and the heating. It is preferable to stir at a moderate speed (based on individual stirrer settings) and heat at a temperature slightly above the boiling point of water (~105-110 °C). Monitor the flask closely (every 2-3 min) for water droplets forming at the bottom of the condenser; when the first (approximate) condensed water droplet falls back in the flask, mark the time as the start of refluxing.
    5. Reflux the CC suspension for ~20 min, or until all the CC has dissolved to generate a 4 mM supersaturated CC solution.
    6. Turn off the stirring and heating.
    7. Remove heating mantle from under the round bottom flask.
      Caution: Both the mantle and the flask will be extremely hot–use of heat-protective gloves and lab coats are strongly recommended.
    8. Let stand for 30-40 min or until the supersaturated CC solution is cooled to almost room temperature, without disturbing the solution.
      Note: Constant water flow through the reflux condenser is still required throughout the process.

  3. Cystine precipitation assay
    1. When the supersaturated CC solution from step B8 is cooled to almost room temperature, carefully detach the Allihn condenser from the flask. Using a pipette transfer 400 μl of this solution to each of the control (positive and negative) as well as the drug vials prepared in steps A9-A11.
      Note: This step should be completed immediately after B8. Also, make sure not to disturb the supersaturated CC solution unnecessarily, i.e., any more than required for the transfer, otherwise CC may crash out.
    2. Briefly vortex each vial to homogenize the contents.
    3. Incubate vials at 4 °C in an incubator or a fridge for 48-72 h (ideally one that maintains consistent temperatures without much fluctuations).
    4. At the end of the incubation period, centrifuge the vials at 1,300 x g at 4 °C for 15 min.
    5. Carefully remove as much of the supernatant from each vial with a 1 ml pipette, without disturbing the crystalline CC precipitate. Next, using gel loading tips and a suitable pipette, remove the remaining supernatant without drawing up any of the precipitated cystine particles. Discard the supernatant.
      Note: This step requires some practice to effectively remove all supernatant while keeping behind most of the CC particles; we recommend a couple of practice runs with the positive and negative controls before running drug experiments.
      Caution: The supernatant may contain hazardous drugs/chemicals; thus disposal of the supernatant should be done according to respective institute guidelines.
    6. Add 3.6 ml of the 30 µM d4-CC solution (prepared in A6) using a 1 ml pipette to each vial (4 x 900 µl transfers to each vial).
    7. Vortex well to combine the contents. The precipitated CC may not dissolve at this stage completely.
    8. Freeze the solutions at -20 °C.
      Notes:
      1. Step C8 is necessary to dissolve the precipitated crystals of CC. In our experience, simply leaving the vials at a -20 °C freezer works best. Attempting to speed up the process by using dry ice or liquid nitrogen results in the glass vial cracking and thus not recommended.
      2. The vials can be stored anywhere between a few hours to several months at -20 °C.

  4. Mass Spectrometry-based quantification of precipitated cystine
    1. When ready for analysis, thaw the contents of the precipitation vials (step C8) at room temperature.
    2. Once at room temperature, vortex the vials briefly and carefully inspect each vial for remaining CC precipitates. Ideally all vials should have clear solutions at this point.
      Note: Following the steps as indicated, we have never experienced residual precipitates at this stage. Nonetheless, it is possible that certain drugs may decrease CC solubility such that a lot of CC precipitates during the incubation and not all go back in solution. In such cases, it will be necessary to transfer the vial contents (including any remaining precipitate) to larger tubes, add more HPLC-grade water (the d4-CC will normalize any dilution effects during mass spectrometric quantification), and redo steps C8 and D1 for these vials.
    3. Transfer 1 ml of the clear solution from each 4 ml vials to prelabeled HPLC autosampler vials.
    4. Prepare a blank vial with 1 ml HPLC-grade water, as well as an internal standard vial with 1 ml of the 30 µM d4-CC solution (prepared in A6).
    5. Analyze samples using HPLC-MS/MS following vendor-specific instructions. Briefly, this comprises the following steps:
      1. Optimize multiple reaction monitoring (MRM) transitions for CC and d4-CC. This includes determination of compound-specific precursor and product ions and optimal MS parameters for each transition (Q1, precursor→Q3, product), achieved by isocratic flow injection of the 10 µM solution for each compound. Label the most intense (Q1→Q3) transition as quantifier and the next best transition as qualifier for each compound (see Table 1).

        Table 1. LC-MS/MS parameters

        Note: DP = Declusturing potential, EP = Entrance potential, CE = Collision energy, and CXP = Collision exit potential.

      2. For LC separation, use a solvent gradient of 95% of 20 mM ammonium acetate (pH ~9.0-9.5) in water and 5% acetonitrile (mobile phase A)–ACN (mobile phase B) at a flow rate of 0.8 ml/min, starting with an acetonitrile content of 55% for 0.25 min, decreased to 2% at 1.15 min and held at 2% until 1.65 min. Reconstitute the LC column subsequently to its initial condition over the next 0.1 min and re-equilibrate for 0.5 min. Under these conditions, CC and d4-CC elute at a retention time = 1.1 min with our HPLC setup. The LC Column is maintained at 35 °C for the analysis.
      3. Operate the mass spectrometer in positive ion mode with the following source conditions (specific to AB Sciex’s 4000 QTRAP instrument): curtain gas (CUR) 20, nebulizer gas (GS1) 30, auxiliary gas (GS2) 30, ionspray voltage (IS) 4,500 V, and source temperature (TEM) 450 °C.
      4. Run the blank sample first, followed by the internal standard sample, and followed by the rest of the assay samples. It is also recommended to run a blank between each type of samples, e.g., blank–4 replicates of Neg control–blank–4 replicates of 50 µM CDME–blank–etc.

        Notes:

        1. Please consult a mass spectrometrist or the University/Institute’s mass spectrometry core for help with these steps.
        2. The steps pertaining to mass spectrometry are provided specifically for use with a triple quadrupole-type instrument. Other mass spectrometers and/or simple scanning m/z of the molecular ions of CC (241) and d4-CC (245), instead of MS/MS detection, may also be used.

Data analysis

  1. Quantify the amount of CC precipitate in each vial by computing the area under the curve (AUC) for the quantifier peak of CC. Figure 1 shows an exemplary AUC, which acts as a surrogate for the CC quantity in the sample. See also Note 3.


    Figure 1. Extracted ion chromatogram (XIC) for the MRM transition (m/z 241.1→152.0) representing cystine (CC). The red shaded area shows the area under the curve (AUC) for CC in this sample, which is proportional to the quantity of CC in the sample.

  2. Normalize the AUC with the corresponding area under the curve for d4-CC peak, to obtain nAUC values. These nAUC values may be used as surrogates for the amount of CC precipitate in each vial.
  3. Compare the normalized negative control nAUC values with that of drug or CDME nAUC values (obtained in step E2), using standard statistical analysis methods, such as Student’s t-test (see Figure 2). CDME is used as a positive control. In our experience, 500 µM CDME results in almost no CC precipitate and very low nAUC, whereas 50 µM CDME results in a moderate amount of precipitate, resulting in modest reduction of nAUC as compared to the blank (Figure 2).
  4. Any drug that results in significant reduction of nAUC values as compared to the blank should be considered for further investigation, such as testing in the mouse model of cystinuria (Zee et al., 2017).


    Figure 2. Relative yield of L-cystine precipitation obtained after crystallization in the presence of water (blank) and L-cystine dimethyl ester (CDME, 50 and 500 µM), a positive control. Error bars are SD.

Notes

  1. We have generally found this assay to be reproducible, so long as the incubation temperature (step 3C) is reasonably consistent between multiple experiments.
  2. Use of HPLC-grade water is highly recommended wherever possible.
  3. D4-CC (3,3,3’,3’-d4-L-cystine), a commercially available isotopolog of cystine (CC) has been used in the protocol to aid in CC estimation. As with most isotopologs, CC and D4-CC are chemically nearly identical; thus, they have similar chemical and physical properties (such as solubility). However, in a mass spectrometry experiment, they are easily distinguishable as CC ionizes at m/z 241 and D4-CC at m/z 245. A specific amount of D4-CC is added to each vial in this experiment–monitoring both CC and D4-CC in the sample helps in understanding if the actual compound (CC) may have undergone any unintended modification or dilution indicated by a change in D4-CC levels.

Recipes

  1. Ammonium acetate solution
    1. Weigh ~3.1 g ammonium acetate in a glass bottle
    2. Add 2 L HPLC water (in small batches to dissolve)
    3. Adjust the pH to 9-9.5 using ammonium hydroxide

Acknowledgments

This work was supported by grants from the American Federation of Aging Research (to P.K.), Larry L. Hillblom Foundation (to P.K.), Boston Scientific Foundation (to M.S.) and the NIH (R01 AG038688 & R01 AG045835 to P.K.; R21 DK091727 to P.K. and M.S.; P20 DK100863 & R21 DE025961 to M.S.). We thank See Yang and David Hall for help with the assay setup. The cystine solubility assay presented here has been developed as part of our recent publication on the identification of α-Lipoic acid as a drug that prevents cystine kidney stones in a mouse model of cystinuria (Zee et al., 2017). We acknowledge the work by Rimer, J. D. et al. in Science (Rimer et al., 2010) that identified CDME to significantly increase cystine solubility and published an earlier version of the assay.

References

  1. Becker, G. and Caring for Australians with Renal, I. (2007). The CARI guidelines. Kidney stones: cystine stones. Nephrology (Carlton) 12 Suppl 1: S4-10.
  2. Dent, C. E. and Senior, B. (1955). Studies on the treatment of cystinuria. Br J Urol 27(4): 317-332.
  3. Koraishy, F. M., Cohen, R. A., Israel, G. M. and Dahl, N. K. (2013). Cystic kidney disease in a patient with systemic toxicity from long-term D-penicillamine use. Am J Kidney Dis 62(4): 806-809.
  4. Mattoo, A. and Goldfarb, D. S. (2008). Cystinuria. Semin Nephrol 28(2): 181-191.
  5. Milliner, D. S. and Murphy, M. E. (1993). Urolithiasis in pediatric patients. Mayo Clin Proc 68(3): 241-248.
  6. Palacin, M., Borsani, G. and Sebastio, G. (2001). The molecular bases of cystinuria and lysinuric protein intolerance. Curr Opin Genet Dev 11(3): 328-335.
  7. Rimer, J. D., An, Z., Zhu, Z., Lee, M. H., Goldfarb, D. S., Wesson, J. A. and Ward, M. D. (2010). Crystal growth inhibitors for the prevention of L-cystine kidney stones through molecular design. Science 330(6002): 337-341.
  8. Saravakos, P., Kokkinou, V. and Giannatos, E. (2014). Cystinuria: current diagnosis and management. Urology 83(4): 693-699.
  9. Sumorok, N. and Goldfarb, D. S. (2013). Update on cystinuria. Curr Opin Nephrol Hypertens 22(4): 427-431.
  10. Zee, T., Bose, N., Zee, J., Beck, J. N., Yang, S., Parihar, J., Yang, M., Damodar, S., Hall, D., O’Leary, M. N., Ramanathan, A., Gerona, R. R., Killilea, D. W., Chi, T., Tischfield, J., Sahota, A., Kahn, A., Stoller, M. L. and Kapahi, P. (2017). α-Lipoic acid treatment prevents cystine urolithiasis in a mouse model of cystinuria. Nat Med 23(3): 288-290.

简介

胱尿蛋白尿是一种罕见的遗传性疾病,其特征在于复发性疼痛的肾结石,主要由胱氨酸,氨基酸半胱氨酸的二聚体组成(Sumorok和Goldfarb,2013)。 使用小鼠模型的胱氨酸尿症,我们最近显示,在尿液中增加胱氨酸溶解度的药物的施用可能是临床治疗疾病的新型治疗策略(Zee等人,2017)。 在该领域需要开发用于开发新药用于胱氨酸尿症的很大需求。 为此,我们在此介绍一种简单的体外胱氨酸溶解度测定法,可用于筛选化合物以鉴定可能影响胱氨酸溶解度的潜在药物。 该测定包括制备胱氨酸过饱和溶液,将该溶液与选择的药物孵育,最后使用高压液相色谱 - 串联质谱(HPLC-MS / MS)定量在各种条件下沉淀的胱氨酸的量。
【背景】胱硫酸尿症是一种肾结石疾病,其特征在于肾脏近端小管中的胱氨酸转运蛋白的遗传缺陷,导致尿液中的胱氨酸负荷大量增加,其作为肾结石沉淀(Sumorok和Goldfarb,2013)。虽然分类为罕见的遗传疾病(约1 / 15,000个人)(Milliner和Murphy,1993; Palacin等人,2001),患有胱氨酸尿症的患者经历复发性石发病的痛苦痛苦(Dent和高级,1955)。与其他更常见的肾结石类型(如草酸钙或尿酸)不同,胱氨酸结石更致密,对体外冲击波碎石术(SWL)有抵抗力,要求患者进行多次急诊室和外科手术以消除梗阻石头(Mattoo和Goldfarb,2008)。目前的胱硫酸尿药物方案适用于增加尿酸(柠檬酸钾)或降低尿胱氨酸水平(硫醇药物,如硫普罗宁),其通常与严重的副作用相关(Koraishy等人,2013; Sumorok和Goldfarb,2013; Saravakos等人,2014)。此外,临床(Becker等人,2007)和鼠模型(Zee等人,2017)研究发现,几乎没有证据表明这些药物最终是有效的,长期患者依从性差。因此,迫切需要开发治疗胱硫酸尿症的有效疗法。我们最近的研究结果表明,增加尿胱氨酸的溶解度可能是一个可行的替代策略药物发展在尿胱磷酸尿。在本文中,我们描述了体外胱氨酸溶解度测定以鉴定能影响胱氨酸溶解度的新化合物。

关键字:胱氨酸尿, 肾结石, 胱氨酸, 质谱法, 药物筛查, L-胱氨酸二甲酯

材料和试剂

  1. 手套
  2. 实验室外套
  3. 培养管(16×100mm)(VWR,目录号:47729-576)
  4. 4毫升玻璃小瓶和盖子(13-425螺纹牙膏,硼硅酸盐玻璃)(VWR,目录号:46610-706)
  5. HPLC自动进样器小瓶和盖子(9-425线程)(VWR,目录号:89523-482)
  6. 移液器吸头(标准1000μl和200μl)
  7. 凝胶装载移液器吸头(VWR,目录号:37001-150)
  8. 将圆底烧瓶连接到Allihn冷凝器的接头夹(VWR,目录号:89426-304)
    制造商:GLASSCOLABS,目录号:007.470.05A。
  9. Eppendorf管(标准1.5 ml)
  10. 乙酸铵(对于LC-MS LiChropur )(EMD Millipore,目录号:533004)
  11. 水(HPLC级)(VWR,BDH ,目录号:BDH23595.400)
  12. 氢氧化铵(H 2 O 2中为28%NH 3,≥99.99%)(Sigma-Aldrich,目录号:338818)
  13. (98%)(剑桥同位素实验室,目录号:DLM) -1000-1)
  14. L-胱氨酸(≥98%)(Sigma-Aldrich,目录号:C8755)
  15. DL-胱氨酸二甲酯二盐酸盐(≥95%)(Sigma-Aldrich,目录号:857327)
  16. 乙腈(HPLC级)(VWR,BDH ,目录号:BDH83639.400)
  17. 乙酸铵溶液(见食谱)

    注意:为所列材料和试剂提供了具体的品牌和目录号(我们的工作中使用的)。但是,我们并没有明确认可任何这些品牌或产品,大多数商品可以从其他知名厂商获得。此外,大多数这些产品/目录号是基于美国目前的可用性,因此其他国家的研究人员可能会发现有必要从另一个供应商处获取材料。

设备

  1. pH计(标准)
  2. 安全护目镜
  3. 化学安全罩
  4. 玻璃瓶(标准500毫升和1升)
  5. 玻璃量筒(标准1升,250毫升和100毫升)
  6. 玻璃圆底烧瓶(标准24/40地面接头,100ml)
  7. 磁力搅拌棒(标准)
  8. 回流装置(标准,由搅拌加热罩组成,具有水套和24/40接头的Allihn冷凝器,自来水源,橡胶管和用于附接的夹子)。遵循供应商的汇编说明
  9. 移液器(标准1000μl和200μl)
  10. 涡旋(标准)
  11. -20°C冰箱(标准)
  12. 4℃培养箱或冰箱(标准)
  13. HPLC-MS系统:
    1. HPLC:具有以下模块的Shimadzu UFLC突出体系(HPLC)(Shimadzu,型号:Prominence UFLC):CBM-20A(通信总线模块),DGU-A 3(脱气器),两个LC-20AD (液相色谱仪,二元泵),SIL-20AC HT(自动进样器)
    2. HPLC柱:Luna NH 2柱(2×50mm,3μm,100)(Phenomenex,目录号:00B-4377-B0)
    3. 质谱仪:配有Turbo V™超声波离子源的AB Sciex 4000 LC-MS / MS质谱仪(AB Sciex,型号:QTRAP),/ / sup> 4000
  14. 离心机(标准品,附有4ml小瓶)
  15. 称重秤(标准)

    注意:标准实验室设备应用于此测定,如图所示。已经为HPLC-MS设置的组件提供了特定的品牌和目录号(如我们的工作中所使用的),这些可以由其他供应商的设备替代;可以说任何HPLC-MS设置都可以用于该测定,但是可能需要根据供应商提供的说明进行额外的优化。

软件

  1. AB Sciex的分析师® v1.6.1,用于数据采集,LC方法的开发和分析物特异性MRM(多反应监测)转换的优化
  2. 对于LC-MS / MS数据分析,AB Sciex的Peakview ® v2.1和Skyline ® v3.6 23

程序

  1. 解决方案的准备和药物标准
    1. 在HPLC级水中准备2升20mM乙酸铵。这将用于HPLC样品制备,以及制备其中一个HPLC流动相。
      注意:2 L是建议体积,根据预期的待测药物数量进行调整。
    2. 通过以约0.5ml的批次加入氢氧化铵然后pH测量将该溶液的pH调节至约9.0-9.5(这不一定是准确或可重复的)。标准pH计可用于此目的;但是,建议将pH探针直接浸入溶液中。相反,将小的等分试样(〜0.5-1ml)转移到培养管中进行每一周的pH测量。
      注意:氢氧化铵腐蚀性很强;在处理该化合物时,应始终佩戴手套,安全护目镜和实验衣。也可以在化学品安全罩内部处理。
    3. 在干净的玻璃瓶(1升)中将950毫升pH调节的20mM乙酸铵溶液测量,并加入50ml乙腈(ACN),使用合适的玻璃瓶。这将用作HPLC的流动相A.
      注意:该溶液可以在室温下储存约2周。瓶子和玻璃瓶应先用HPLC水彻底清洗,然后用ACN彻底清洗(使用清洁剂/肥皂)。
    4. 测量1 L的ACN到另一个干净的玻璃瓶(1升)。这将用作HPLC的流动相B。
      注意:ACN可以在室温黑暗中储存,使用时间长达6个月。
    5. 将400ml pH调节的20mM乙酸铵溶液(来自步骤A2)置于500ml玻璃瓶中。重量约29.2mg的3,3,3',3' - -L-胱氨酸(称为 d 4 -CC),并充分混合并溶解并生成300μM EM> 4 -CC。
      注意:该解决方案可以在-20°C下等分并储存几个月。
    6. 从这个300μM的解决方案中准备足够量的30μM通过用pH调节的20mM乙酸铵溶液(来自步骤A2)稀释(10倍)来进行。
      注意:此解决方案应为每批准备新鲜。 3.6ml该溶液将作为测定中使用的每个小瓶的内标使用,根据被测试的药物(和复制)的数量计算所需的量。
    7. 在HPLC级水(5x)中准备500μl各2.5mM和250μMDL-胱氨酸二甲酯(CDME)溶液。使用CDME作为测定的阳性对照,因为已显示其显着增加胱氨酸的溶解度(Rimer等人,2010; Zee等人,2017)。
    8. 准备试验药物溶液(5x):称量测试药物并溶解在HPLC级水中以达到比预期要高5倍的浓度;在测定过程中,将其稀释至1倍。
      注意:药物可能会造成健康和/或环境危害;因此,应以妥善的安全预防措施处理。
    9. 准备阳性对照小瓶(每个4个重复):两套4 x 4毫升玻璃小瓶,每个100微升2.5微克和250微克CDME溶液(来自步骤A7)。在测定过程中,这将被稀释5倍,以达到最终浓度分别为500μM和50μMCDME。
    10. 准备阴性对照小瓶(每个4个重复):一套4×4ml玻璃小瓶,每个HPLC水100μl。
    11. 准备药瓶(每个4个重复):一套4×4ml玻璃小瓶,每个5μl药物溶液100μl(来自步骤A8)。在测定过程中,将其稀释至1倍。

      注意:

      1. 对于步骤A9-A11,应使用每个条件至少3次重复(理想情况下4次重复)。
      2. 所示的硼硅酸盐玻璃小瓶对于该步骤绝对是必需的;塑料小瓶与此测定不兼容

  2. 制备胱氨酸过饱和溶液
    1. 在圆底烧瓶中加入〜96mg的L-胱氨酸(称为CC),并加入100ml的HPLC级水,以达到终浓度为4mM的CC。
      注意:
      1. 再次用HPLC级水,ACN和水彻底清洗圆底烧瓶。避免使用肥皂/洗涤剂。
      2. CC在这个阶段不会解散。
      3. 100毫升是一个建议的体积,根据预期的药物数量进行调整。这是一个典型的情况,当10种药物被测试在两个浓度(每个条件4个复制):在这种情况下,将有80个药物小瓶(10药物x 2浓度x 4重复每个)和额外的12个小瓶为阳性和每个设置中使用的阴性对照(参见步骤A9和A10),即总共92×4ml小瓶。对于这样的设置,需要总共92×400μl,即36.8ml的4mM CC溶液(参见下文,步骤C1)。因此,制备50ml回流溶液(通过称重〜48mg CC)就足够了。
    2. 加入一个磁力搅拌棒(适合尺寸)。
    3. 将装有搅拌棒和CC悬浮液的圆底烧瓶装入回流装置。使用夹子将烧瓶牢固地连接到Allihn冷凝器,并打开通过冷凝器护套的水流。确保通过冷凝器达到稳定(但不是活力)的水流。
      注意:回流是指将溶剂(或悬浮液)加热至沸点并允许蒸发的溶剂冷凝回原始烧瓶的过程,最常用冷却水流过Allihn冷凝器。回流确保溶剂(或悬浮液)始终保持在其沸点(固定温度),长时间,而不会由于蒸发造成任何显着的溶剂损失。
      注意:回流装置应安装在化学品安全罩内。该过程需要在高温下加热,因此应采取适当的安全预防措施。
    4. 当通过回流冷凝器的水流稳定时,开启搅拌和加热。优选以中等速度(基于各个搅拌器设置)并在略高于水沸点(〜105-110℃)的温度下加热。仔细观察烧瓶(每2-3分钟一次),使冷凝器底部形成水滴;当第一(近似)冷凝水滴落回烧瓶中时,将时间标记为开始回流。
    5. 将CC悬浮液回流约20分钟,或直到所有CC溶解以产生4mM过饱和CC溶液。
    6. 关闭搅拌和加热。
    7. 从圆底烧瓶中取出加热套。
      注意:强烈建议地幔和烧瓶都非常热,使用防护手套和实验室外套。
    8. 放置30-40分钟,或直到过饱和CC溶液冷却至几乎室温,而不影响溶液。
      注意:在整个过程中仍然需要恒定的水流过回流冷凝器。

  3. 胱氨酸沉淀试验
    1. 当将来自步骤B8的过饱和CC溶液冷却至几乎室温时,小心地将Allihn冷凝器从烧瓶中分离出来。使用移液管将400μl该溶液转移到对照(阳性和阴性)以及步骤A9-A11中制备的药物瓶中。
      注意:此步骤应在B8之后立即完成。此外,请确保不要不必要地干扰过饱和CC解决方案,即不仅需要传输,否则CC可能会崩溃。
    2. 简单地旋转每个小瓶以使内容物均质化。
    3. 在4℃的孵育箱或冰箱中孵育小瓶48-72小时(理想情况下保持一致的温度,没有太大的波动)。
    4. 在孵育期结束时,在4℃下以1,300×g离心小瓶15分钟。
    5. 用1 ml移液管小心地清除每个小瓶中的上清液,而不会影响结晶CC沉淀。接下来,使用凝胶加载尖端和合适的移液管,除去剩余的上清液,而不会吸收任何沉淀的胱氨酸颗粒。丢弃上清液。
      注意:该步骤需要一些实践来有效地去除所有上清液,同时保留大部分CC颗粒;我们建议在运行药物实验之前,先进行一些正面和负面对照的练习。
      注意:上清液中可能含有危险药物/化学物质;因此上清液的处理应按照各自的研究方针进行。
    6. 使用1ml移液管向每个小瓶(4×900)加入3.6ml30μM的细胞-CC溶液(在A6中制备) μl转移到每个小瓶)
    7. 涡旋很好地结合内容。沉淀的CC可能在此阶段完全溶解。
    8. -20℃冷冻溶液。
      注意:
      1. 步骤C8需要溶解沉淀的CC晶体。根据我们的经验,只需将小瓶放在-20°C的冷冻室即可。试图通过使用干冰或液氮加速过程,导致玻璃小瓶开裂,因此不推荐。
      2. 这个小瓶可以储存在-20°C的几个月到几个月之间的任何地方
  4. 沉淀胱氨酸的基于质谱的定量
    1. 当准备好分析时,在室温下解冻沉淀小瓶的内容物(步骤C8)。
    2. 一旦在室温下,短暂旋转小瓶,仔细检查每个小瓶以保留CC沉淀物。在这一点上,所有的小瓶都应该有明确的解决方案。
      注意:按照指示的步骤,我们在这个阶段从未经历过残留的沉淀。尽管如此,某些药物可能会降低CC溶解度,使得大量的CC在孵育期间沉淀,而不是全部回到溶液中。在这种情况下,有必要将小瓶内容物(包括任何剩余的沉淀物)转移到较大的管中,加入更多的HPLC级水(4/6/6) -CC将在质谱定量过程中对任何稀释效应进行归一化),并为这些小瓶重做步骤C8和D1。
    3. 将1毫升透明溶液从每4毫升小瓶转移到预标记的HPLC自动进样器小瓶中。
    4. 用1ml HPLC级水准备一个空白的小瓶,以及一个内径标准的小瓶,其中有1 ml的30μM CC溶液(在A6中制备)。
    5. 按照供应商特定的说明,使用HPLC-MS / MS分析样品。简而言之,这包括以下步骤:
      1. 优化CC和MD的多重反应监测(MRM)转换 4 /子> -CC。这包括化合物特异性前体和产物离子的测定以及每个过渡的最佳MS参数(Q 1 ,前体→ Q 3 ,产品),通过等度流注入为每个化合物10μM解决方案。标签最强(Q 1 →Q 3 )转换为量词,并将下一个最佳转换作为每个化合物的限定符(见表1)。

        表1. LC-MS / MS参数

        注意:DP =去离子电位,EP =入口电位,CE =碰撞能量和CXP =碰撞出口电位。

      2. 对于LC分离,使用95%的20mM乙酸铵(pH〜9.0-9.5)在水和5%乙腈(流动相A)-ACN(流动相B)中的溶剂梯度,流速为0.8ml / min ,乙腈含量为55%0.25分钟,在1.15分钟下降至2%,保持在2%至1.65分钟。随后在随后的0.1分钟内将LC柱重建为初始条件,并重新平衡0.5分钟。在这些条件下,使用我们的HPLC设置,CC和 LC柱保持在35℃进行分析。
      3. 使用以下源条件(AB Sciex的4000 QTRAP仪器专用)操作质谱仪:帘式气体(CUR)20,喷雾器气体(GS1)30,辅助气体(GS2)30,离子喷射电压(IS)4,500 V和源温度(TEM)450℃。
      4. 首先运行空白样品,然后是内标样品,然后是其余的测定样品。还建议在每种类型的样品之间运行空白,例如,50μMCDME-blank-em等的Neg-control-blank-4重复的空白-4复制。

        注意:

        1. 请咨询质谱仪或大学/研究所的质谱核心,以获得有关这些步骤的帮助。
        2. 与质谱有关的步骤专门用于三重四极杆型仪器。 CC(241)和d 分子离子的其他质谱仪和/或简单扫描m / z, em> -CC(245),也可以使用MS / MS检测。

数据分析

  1. 通过计算CC的量词峰值曲线下面积(AUC)来量化每个小瓶中CC沉淀物的量。图1示出了示例性AUC,其用作样品中CC量的替代物。另见注3

    图1.表示胱氨酸(CC)的MRM转变的提取离子色谱图(XIC)( m / z 241.1→152.0) 。 红色阴影区域显示该样品中CC的曲线下面积(AUC),与样品中CC的数量成正比。

  2. 使用曲线下的对应区域归一化AUC,以获得nAUC值,以获得nAUC值。这些nAUC值可以用作每个小瓶中CC沉淀量的替代物。
  3. 使用标准统计分析方法,例如Student's试验(参见图2),比较归一化的阴性对照nAUC值与药物或CDME nAUC值(在步骤E2中获得)的值。 CDME用作阳性对照。在我们的经验中,500μMCDME几乎没有CC沉淀和非常低的nAUC,而50μMCDME导致中等量的沉淀,导致与空白相比nAUC的适度降低(图2)。
  4. 与空白相比,导致nAUC值显着降低的任何药物都应考虑进一步研究,例如在胱氨酸尿的小鼠模型中测试(Zee等人,2017)。


    图2.在水(空白)和L-胱氨酸二甲酯(CDME,50和500μM)存在下结晶后获得的L-胱氨酸沉淀的相对产率,阳性对照。 错误栏是SD。

笔记

  1. 我们通常认为这种测定是可重复的,只要培养温度(步骤3C)在多个实验之间是相当一致的。
  2. 尽可能高度推荐使用HPLC级水。
  3. D4-CC(3,3,3',3' - 4 -L-胱氨酸),市售的胱氨酸同位素( CC)已经用于协议以帮助CC估计。与大多数同位素相似,CC和D4-CC在化学上几乎相同;因此,它们具有相似的化学和物理性质(如溶解度)。然而,在质谱实验中,它们容易区分,因为在m / z 241处的CC电离和在m / z 245处的D4-CC。在该实验中,每个小瓶中加入特定量的D4-CC,监测样品中的CC和D4-CC有助于了解实际化合物(CC)是否可能经历D4中变化所指示的任何意外修改或稀释-CC水平。

食谱

  1. 乙酸铵溶液
    1. 称量〜3.1g乙酸铵在玻璃瓶中
    2. 加入2升HPLC水(小批量溶解)
    3. 使用氢氧化铵将pH调节至9-9.5

致谢

这项工作得到了美国老龄化研究联合会(PK),Larry L. Hillblom基金会(PK),波士顿科学基金会(MS)和NIH(R01 AG038688& R01 AG045835至PK; R21 DK091727)的资助。至PK和MS; P20 DK100863& R21 DE025961至MS)。感谢见杨和大卫·霍尔为检测设置提供帮助。这里提出的胱氨酸溶解度测定作为我们最近出版的关于α-硫辛酸作为在胱氨酸尿症小鼠模型中预防胱氨酸肾结石的药物的鉴定的一部分而开发的(Zee等人, 2017年)。我们承认Rimer,J.D。等人的工作。确定CDME以显着增加胱氨酸溶解度并发表了早期版本的测定法的Science(Rimer et al。,2010)中的。

参考

  1. Becker,G.和“关心澳大利亚人与Renal”(2007)。 CARI指南。肾结石:胱氨酸石。 肾脏科(Carlton)<12>增补1:S4-10。
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  3. Koraishy,FM,Cohen,RA,Israel,GM和Dahl,NK(2013)。&nbsp; 来自长期D-青霉胺使用的全身毒性患者的囊性肾脏疾病。 Am J Kidney Dis 62(4):806-809。 br />
  4. Mattoo,A.and Goldfarb,DS(2008)。&nbsp; 半胱氨酸尿症。 Semin Nephrol 28(2):181-191。
  5. Milliner,DS and Murphy,ME(1993)。&nbsp; 尿石症在儿科患者中。 Mayo Clin Proc 68(3):241-248。
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引用:Bose, N., Zee, T., Kapahi, P. and Stoller, M. L. (2017). Mass Spectrometry-based in vitro Assay to Identify Drugs that Influence Cystine Solubility. Bio-protocol 7(14): e2417. DOI: 10.21769/BioProtoc.2417.
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