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Extraction and Analysis of Pan-metabolome Polar Metabolites by Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS)
超高效液相色谱 - 串联质谱法(UPLC-MS/MS)提取和分析泛代谢组极性代谢产物   

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Cell Metabolism
Apr 2017

Abstract

Modern triple quadrupole mass spectrometers provide the ability to detect and quantify a large number of metabolites using tandem mass spectrometry (MS/MS). Liquid chromatography (LC) is advantageous, as it does not require derivatization procedures and a large diversity in physiochemical characteristics of analytes can be accommodated through a variety of column chemistries. Recently, the comprehensive optimization of LC-MS metabolomics using design of experiments (COLMeD) approach has been described and used by our group to develop robust LC-MS workflows (Rhoades and Weljie, 2016). The optimized LC-MS/MS method described here has been utilized extensively for metabolomics analysis of polar metabolites. Typically, tissue or biofluid samples are extracted using a modified Bligh-Dyer protocol (Bligh and Dyer, 1959; Tambellini et al., 2013). The protocol described herein describes this workflow using targeted polar metabolite multiple reaction monitoring (MRM) from tissues and biofluids via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This workflow has been utilized extensively for chronometabolic analysis (Krishnaiah et al., 2017), with applications generalized to other types of analyses as well (Sengupta et al., 2017; Sivanand et al., 2017).

Keywords: UPLC-MS (UPLC-MS), Tandem mass spectrometry (串联质谱法), Metabolomics (代谢组学), Triple quadrupole (三重四极杆), Liquid-liquid extraction (液-液萃取), MRM (MRM), Liquid chromatography (液相色谱法)

Background

Metabolomics is a field of study aiming to comprehensively analyze metabolites through the use of various analytical detection methods, namely mass spectrometry (MS) and nuclear magnetic resonance (NMR) (Liu and Locasale, 2017). Although both NMR and MS are essential tools in metabolomics, mass spectrometry can analyze samples with greater sensitivity (Liu and Locasale, 2017). Within mass spectrometry, various approaches are available, however a rapid approach to profile the global metabolome is needed (Lv et al., 2011; Rhoades and Weljie, 2016). Advancements in triple quadrupole mass spectrometers have made them well suited for ion-switching methods (scanning in both positive and negative ion modes within a single analysis) in addition to enabling reproducible and sensitive targeted profiling of numerous metabolites (Lv et al., 2011; Gika et al., 2012; Yuan et al., 2012; Rhoades and Weljie, 2016). Additionally, LC typically does not require extensive sample preparation, nor derivatization, which allows for the detection of a broader range of metabolites (Gika et al., 2012; Liu and Locasale, 2017). Nonetheless, development of LC-MS methods to comprehensively analyze small polar metabolites is nontrivial, and requires advanced modeling to optimize both LC and MS factors simultaneously. Thus, the COLMeD approach aimed to address this challenge and enabled a more comprehensive metabolite analysis across platforms including on a triple quadrupole mass spectrometer (Rhoades and Weljie, 2016). The LC-MS workflow on a triple quadrupole is described here and has been used to successfully study metabolomics in a circadian context in which 179 metabolites were successfully profiled and analyzed (Krishnaiah et al., 2017).

Materials and Reagents

  1. Pipette tips 1,000 μl, 200 μl, 10 μl (Gilson, catalog numbers: F1735001 , F1733001 , F1732001 )
  2. 1.7 ml PosiClick tubes (Denville Scientific, catalog number: C2170 )
  3. 2.0 ml Safe-Lock tubes (Eppendorf, catalog number: 022363352 )
  4. Mass spectrometry vials and caps–Verex Vial Kit, 9 mm, PP, 300 μl + PTFE/Silicone, pre slit (Phenomenex, catalog number: AR0-9992-13 )
  5. Stainless steel beads 5 mm (QIAGEN, catalog number: 69989 )
  6. Gloves (Denville Scientific, catalog number: G4161 )
  7. Sample trays
  8. VanGuard cartridge holder (WATERS, catalog number: 186007949 )
  9. XBridge BEH Amide 2.5 μm XP Vanguard Cartridge, 2.1 x 5 mm (WATERS, catalog number: 186007763 )
  10. XBridge BEH Amide 2.5 μm, 2.1 x 100 mm column XP (WATERS, catalog number: 186006091 )
  11. Acquity UPLC column In-Line Filter Kit (WATERS, catalog number: 205000343 )
  12. MilliQ water–18 mΩ, 0.22 μm filter (Merck, catalog number: MPGP04001 )
  13. Acetonitrile–Optima LC/MS (Fisher Scientific, catalog number: A955-4 )
  14. Ammonium acetate (Sigma-Aldrich, catalog number: 73594-25G-F )
  15. Ammonium hydroxide TraceMetal grade (Fisher Scientific, catalog number: A512-P500 )
  16. Argon compressed gas (Airgas)
  17. Chloroform (Fisher Scientific, catalog number: C298-1 )
  18. Methanol optima LCMS (Fisher Scientific, catalog number: A456-4 )
  19. Nitrogen gas (Airgas, catalog number: NI230LT230RB )
  20. Solvent A (see Recipes)
  21. Solvent B (see Recipes)
  22. Seal wash (see Recipes)

Equipment

  1. Pipettes (P1000, P200, P20, P10)
  2. Centrifuge (Eppendorf, model: 5430 R , catalog number: 022620511)
  3. Vortex
  4. ACQUITY H-Class UPLC (WATERS, model: ACQUITY UPLC H-Class )
  5. Bath Sonicator (VWR, catalog number: 97043-976 )
  6. Speed Vacuum–Vacufuge Plus (Eppendorf, model: Vacufuge plus , catalog number: 022822993)
  7. TissueLyser II (QIAGEN, catalog number: 85300 )
  8. Xevo TQ-S Micro (WATERS, model: Xevo TQ-S Micro )

Software

  1. MassLynx Version 4.1
  2. TargetLynx XS
  3. R (version 3.3)

Procedure

  1. Extracting polar metabolites
    Polar metabolites were extracted using a modified Bligh-Dyer protocol as described below (Bligh and Dyer, 1959; Tambellini et al., 2013). Extractions were performed on ice unless otherwise indicated.
    1. One set of 1.5 ml microfuge tubes for the collection of polar metabolites should be labeled for each sample. (A second set can be labeled if collection of the nonpolar metabolites is desired as well.) The number of biological replicates required for analysis is dependent upon the study and can range from as few as 5 for highly controlled models to several hundred to thousands for human studies.
    2. The following solvents should be prepared and cooled prior to beginning the extraction protocol:
      1. 2:1 Methanol:Chloroform mixture (300 μl per sample)
      2. Pure methanol (100 μl per sample)
      3. Water (18 mΩ, 0.22 μm filtered) (100 μl per sample)
    3. Centrifuge should also be cooled to 4 °C.
    4. Samples (i.e., U2OS and primary hepatocyte cell pellets [number of cells equivalent to a 50 mg cell pellet] or 50 mg of liver tissue) should be placed on ice to thaw.
      Notes:
      1. Smaller sample sizes can be successfully used. E.g., 1 x 106 U2OS cells were utilized in Krishnaiah et al., 2017.
      2. If extracting tissue, 2 ml safe-lock microfuge tubes should be utilized.
    5. (Optional) If quantitation is required, stable-isotope labeled internal standards at a concentration determined on a per metabolite basis should be added to thawed samples at this point.
    6. Once samples are thawed, follow Step A6a if extracting cell pellets or A6b if extracting tissue.
      1. Extraction of cell pellets:
        1. To each sample, 300 μl of the cold 2:1 Methanol:Chloroform mixture is added.
        2. Each microfuge tube containing the sample is vortexed until a homogenous mixture is formed. Figure 1A shows a representative endpoint.
          Note: The cell pellet may not be homogenized upon vortexing and thus it is essential to sonicate the sample.
        3. Next, the samples are sonicated in a bath sonicator for 15 min at 35 kHz. Figure 1B shows a representative endpoint.
        4. At the end of sonication, samples are placed back on ice.


          Figure 1. Examples of representative endpoints during an extraction. A. An example of a cell pellet extraction to which 300 μl of 2:1 Methanol:Chloroform was added and vortexed. B. An example of a cell extraction after sonication (Step A6a.iii). C. The aqueous (1) and organic (2) layers are shown at the end of centrifugation (Steps A10 to A11).

      2. Extraction of tissue using the TissueLyser II:
        1. Pre-cool tissue homogenizer blocks by placing at -80 °C.
        2. To each tissue sample, carefully add a stainless steel bead followed by 300 μl of the cold 2:1 Methanol:Chloroform mixture.
        3. Next, using the pre-cooled blocks (handle using gloves), homogenize the tissue samples at 25 Hz for 4 min using the TissueLyser II.
        4. At the end of homogenization, place the samples back on ice.
    7. Next, 100 μl each of water and chloroform are added to each microfuge tube containing the samples (cell pellets or tissue).
    8. The samples are then vortexed again until a homogenous mixture is formed.
    9. In order to separate the polar and non-polar metabolites, microfuge tubes containing the samples are centrifuged at 18,787 x g (RCF) for 7 min at 4 °C.
    10. Samples are then placed back on ice carefully in order to avoid disruption of the separated layers.
    11. Next, carefully collect the upper layer (indicated as layer 1 in Figure 1C) containing the polar metabolites into the pre-labeled set of microfuge tubes taking care not to disturb the interface and/or collect insoluble particulates. (The lower fraction [indicated as layer 2 in Figure 1C] containing nonpolar metabolites can be collected in a similar manner if desired.)
      Note: It is recommended to perform the extraction procedure on test samples prior to actual samples.
    12. The microfuge tubes containing the aqueous layer metabolites are then dried in a speed vacuum for 4 h or until dry.
    13. Upon completion of drying, metabolites can either be re-suspended as indicated below or stored at -80 °C.
      Note: Storing dried samples at -80 °C impacts the stability of metabolites in a differential manner dependent on various compound classes, therefore, it is recommended to perform LC-MS analysis soon after extraction.

  2. Re-suspending and preparing polar metabolites for LC-MS
    1. A randomized run order should be generated in order to minimize variation as a result of sample preparation order.
    2. A minimum of 5 blanks in addition to 10 equilibration (EQ) samples will be run at the beginning of the analysis (see Step B13 in this section for information on preparing EQ and QC samples). Next, 4 quality control (QC) samples will be run followed by the samples. Every 6 to 10 samples, a QC will be run in addition to 2 QC’s at the end of the analysis (Figure 2). Note that the spacing between QC injections can vary, and may be dependent on sample quantity. Greater QC frequency (e.g., every 6 samples) provides more robust data correction, however requires more sample.


      Figure 2. An example of a sample run order is shown. At least 5 blanks (blue) followed by 10 equilibration samples (green) should be run at the start of an analysis. This is followed by 4 quality control samples (red), samples to be analyzed (purple), and then 2 quality control samples again at the end. The example shows a quality control sample run every 6 samples throughout the analysis, however, this can vary from every 6 to 10 samples.

    3. Diluent consisting of 1:1 Acetonitrile:Water should be prepared and cooled beforehand.
    4. In addition, the centrifuge should be cooled to 4 °C.
    5. Dried polar metabolites, diluent as well as sample tray(s) should be placed on ice.
    6. 100 μl of diluent are added to each microfuge tube containing the dried polar metabolites as well as to an empty microfuge tube to be used as a blank. Again, the volume metabolites are resuspended in is dependent on the sample concentration and is adjusted to ensure metabolites are detected within a linear range. Proper dilution of metabolites also ensures longer column life.
    7. Each microfuge tube is then vortexed for a minimum of 20 sec in order to re-suspend the metabolites.
    8. Next, centrifuge the microfuge tubes for 7 min at 18,787 x g (RCF) at 4 °C as an additional precautionary step to clean up samples.
    9. The supernatant from each sample is then aliquoted as follows:
      1. 5 μl is added to a microfuge tube to generate a pooled quality control (QC) sample. (Volume added to the pooled quality control sample is dependent on the total number of quality control samples to be run. For instance, if 10 QC samples are to be run, a minimum of 200 μl would be required if 20 μl are to be added to each vial.)
      2. A minimum of 20 μl is added to LC-MS vials for each replicate to be analyzed. (A minimum of 2 analytical replicates, ideally 3 is recommended.) Volume added to each vial is dependent on injection volume as injection volumes can range from 2 to 10 μl. (It has been noted that smaller injection volumes yield better chromatography.)
        Note: It should be noted that MS vials with inserts were utilized and larger volumes may be required if utilizing standard 1.5 ml HPLC vials.
    10. LC-MS vials are placed in sample tray(s) according to a pre-randomized run order.
    11. 90 μl of the blank (or volume sufficient for the number of blanks to be run) is added to an LC-MS vial and placed in the sample tray.
    12. Once all samples have been aliquoted, the pooled QC sample is vortexed to ensure homogeneity.
    13. From the pooled QC sample:
      1. An equilibration sample is prepared by diluting the QC sample 10 fold using the diluent (1:1 Acetonitrile:Water).
      2. 20 μl is added to mass spec vials and vials are placed in the appropriate positions in the sample tray(s). (The number of vials is dependent on total QC samples to be run and the volume added to vials varies based on desired injection volume.)
    14. 90 μl of the EQ sample is added to an LC-MS vial and placed in the sample tray. Volume can be adjusted based on the number of EQ’s to be run.

  3. Acquiring data by UPLC-MS
    Polar metabolites were then analyzed through UPLC-MS utilizing a method optimized through the COLMeD approach as described in (Rhoades and Weljie, 2016). The following procedure should be used as guidelines in acquiring data by individuals trained in using a mass spectrometer.
    1. Solvent A is prepared fresh according to Recipe 1. In addition, ensure that there is enough of solvent B (Recipe 2) and the seal wash (Recipe 3) present. If not prepare the solvents.
    2. An XBridge BEH Amide column (2.5 μm 100 x 2.1 mm) with a pre-column inline filter and vanguard is utilized at a temperature of 40 °C. The column is utilized on an Acquity (H-Class) UPLC System coupled to a Waters Xevo Triple quadrupole mass spectrometer.
    3. The temperature for the sample manager is set to 8 °C.
    4. Additionally, for the duration of the analysis, nitrogen pressure should be maintained above 100 PSI. Argon gas is used as the collision gas.
    5. Solvent lines, seal wash, and sample manager are primed prior to the start of the analysis. In addition, the needle is washed using 100% acetonitrile. The mass spectrometer is placed in operation for a minimum of 30 min prior to the start of the analysis.
    6. The column is equilibrated in the solvents as well as the starting conditions prior to beginning the analysis according to Water’s XP 2.5 micron Columns Care and Use Manual.
    7. The following parameters as indicated in Table 1 are utilized for the LC Inlet method for each injection. The run time for each injection is 30 min followed by a 1 min seal wash.

      Table 1. LC parameters


    8. Parameters utilized for the mass spectrometer in MSMS mode are indicated in Table 2 and are utilized for both electrospray positive and negative modes.

      Table 2. MS parameters for both positive and negative modes


    9. 2 μl of the blanks and equilibration samples are injected while a 2 to 10 μl injection volume is utilized for the quality control samples and samples.
    10. Samples are analyzed utilizing a multiple reaction monitoring (MRMs) mode in which 338 metabolites are targeted through their mass over charge ratios however the number of metabolites detected varies based on sample concentration, sample type, as well as sample handling. MRMs were generated utilizing the databases METLIN (Smith et al., 2005) and HMDB (Wishart et al., 2013) in addition to Waters’ IntelliStart software and standards as well as prior established protocols (Basu and Blair, 2011; Yuan et al., 2012). Metabolites scanned for and their settings (transitions and cone and collision voltages) are detailed in Table S1.

Data analysis

  1. Data acquired through LC-MS as indicated above is processed through the use of the TargetLynx software. This software is utilized to identify peaks from Total Intensity Chromatograms, integrate them and to obtain ion counts (Figure 3).


    Figure 3. An example of a total intensity chromatogram is shown. Here, the peak for methionine was identified and integrated using TargetLynx resulting in an ion count of 994753.69.

  2. Ion counts obtained from TargetLynx are then exported to a text file for further processing in R.
  3. In R, a customized script is utilized to remove metabolites with metabolic features appearing in less than 50% of QC samples or those that have a coefficient of variation greater than 30%. The QC samples are also used to fit a cross-validated locally estimated scatterplot smoothing function (LOESS) to each metabolite (Dunn et al., 2011) in order to account for instrumental drift over the course of the analysis to be used for normalization of ion counts (Figure 4). (The R script is available upon request however it should be noted that it is specific for data obtained using Waters’ TargetLynx software.)
  4. The normalized ion counts are then utilized for further statistical and multivariate analyses.


    Figure 4. Principal Component Analysis (PCA) of a sample data set is shown. A PCA of the average of 2 analytical replicates for 2 Drosophila genotypes (Fumin and Sleepless) shows the clustering of quality control (QC) samples at various steps in the data analysis process. QC samples (red) are shown prior to LOESS correction and Normalization (A) to exhibit a greater spread as compared to after LOESS Correction (B). QC samples remain tightly clustered after median fold change normalization of the LOESS corrected Data (C).

Notes

The above described extraction protocol has been utilized in the analysis of cell pellets (Krishnaiah et al., 2017), liver (Krishnaiah et al., 2017), whole Drosophila (Maguire et al., 2015), and brain tissues (McGrath et al., 2008) as well as other sample types such as plasma (Banoei et al., 2017), serum (Beaudry et al., 2016 and Hao et al., 2016), cereberal spinal fluid biofluids, yeast (Tambellini et al., 2013 and 2017) etc. We also strongly recommend that any results be validated by inclusion of a stable-isotope labeled internal standard prior to extraction or unlabeled standard analyzed in a similar matrix to confirm retention times and fragmentation patterns.

Recipes

  1. Solvent A
    95:5 Water:Acetonitrile
    20 mM ammonium acetate
    Ammonium hydroxide, pH of 9
    Sonicate at 35 kHz for 15 min
  2. Solvent B
    100% acetonitrile
    Sonicate for 5 min
  3. Seal wash
    50:50 Water:Acetonitrile
    Sonicate at 35 kHz for 15 min

Acknowledgments

D.M.M and S.D.R. are supported through a Pharmacology T32 Training Grant (T32 GM008076) and the project was supported in part by the Institute for Translational Medicine and Therapeutics’ (ITMAT) Transdisciplinary Program in Translational Medicine and Therapeutics of the University of Pennsylvania via an award to AMW. The project described was supported by the National Center for Research Resources, Grant UL1RR024134, and is now at the National Center for Advancing Translational Sciences, Grant UL1TR000003. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The above-described protocol has been adapted from (Rhoades and Weljie, 2016) and (Krishnaiah et al., 2017). The authors declare no conflicts of interest or competing interests.

References

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  2. Basu, S. S. and Blair, I. A. (2011). SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards. Nat Protoc 7(1): 1-12.
  3. Beaudry, P., Campbell, M., Dang, N. H., Wen, J., Blote, K., and Weljie, A. M. (2016). A pilot study on the utility of serum metabolomics in neuroblastoma patients and xenograft models. Pediatr Blood Cancer 63(2): 214-220.
  4. Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8): 911-917.
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  7. Hao, D., Sarfaraz, M. O., Farshidfar, F., Bebb, D. G., Lee, C. Y., Card, C. M., David, M., and Weljie, A. M. (2016). Temporal characterization of serum metabolite signatures in lung cancer patients undergoing treatment. Metabolomics 12: 58.
  8. Krishnaiah, S. Y., Wu, G., Altman, B. J., Growe, J., Rhoades, S. D., Coldren, F., Venkataraman, A., Olarerin-George, A. O., Francey, L. J., Mukherjee, S., Girish, S., Selby, C. P., Cal, S., Er, U., Sianati, B., Sengupta, A., Anafi, R. C., Kavakli, I. H., Sancar, A., Baur, J. A., Dang, C. V., Hogenesch, J. B. and Weljie, A. M. (2017). Clock regulation of metabolites reveals coupling between transcription and metabolism. Cell Metab 25(4): 961-974 e964.
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  12. McGrath, B. M., McKay, R., Dave, S., Seres, P., Weljie, A. M., Slupsky, C. M., Hanstock, C. C., Greenshaw, A. J., and Silverstone, P. H. (2008). Acute dextro-amphetamine administration does not alter brain myo-inositol levels in humans and animals: MRS investigations at 3 and 18.8 T. Neurosci Res 61(4): 351-359.
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简介

现代三重四极杆质谱仪提供检测和定量大量使用串联质谱(MS / MS)的代谢物的能力。液相色谱(LC)是有利的,因为它不需要大量的生理化学特性。近日,采用的实验(Colmed)的方式设计LC-MS代谢组的全面优化已经被描述和使用我们集团发展稳健的LC-MS工作流程(罗兹和Weljie,2016)。优化的LC-MS / MS方法已被广泛应用于极性代谢物的代谢组学分析。典型地,组织或生物流体样品使用经修饰的Bligh-Dyer的协议萃取(Bligh和Dyer的,1959; Tambellini 等人,2013年)。在此描述的方案使用靶向极性代谢物从通过超高效液相色谱 - 串联质谱法(UPLC-MS / MS)的组织和生物流体多反应监测(MRM)描述的工作流程。此工作流已被广泛用于chronometabolic分析(Krishnaiah 等人,第二千〇十七)中,用推广到其他类型的分析,以及应用程序(森古普塔等人 2017。 Sivanand等人,2017)。


【背景】质谱(MS)和核磁共振(NMR)(Liu和Locasale,2017)。虽然核磁共振和质谱是代谢组学的重要工具,但质谱可以更灵敏地分析样品(Liu and Locasale,2017)。在质谱法中,有多种方法可用,但是需要快速方法来描述全球代谢组学(Lv et al。,2011,Rhoades and Weljie,2016)。三重四极杆质谱仪的进步也已经被用于离子切换方法,此外还能够对许多代谢物进行可重复和灵敏的靶向分析(Lv et al。 ,2011,Gika et al。,2012,Yuan等人,2012; Rhoades and Weljie,2016)。另外,LC典型地不需要大量的样品制备,也不需要衍生作用,这允许检测更广泛的代谢物(Gika等人,2012; Liu和Locasale,2017)。尽管如此,开发用于全面分析小极性代谢物的LC-MS方法是非平凡的,并且同时需要同时建模和优化LC和MS因子。因此,COLMeD方法旨在解决这一挑战,并提供包括三重四极质谱仪(Rhoades和Weljie,2016)在内的跨平台的更全面的代谢物分析。在三重四极杆上的LC-MS工作流程已经在昼夜环境中成功地进行了分析和分析(Krishnaiah等人,2017)。

关键字:UPLC-MS, 串联质谱法, 代谢组学, 三重四极杆, 液-液萃取, MRM, 液相色谱法

材料和试剂

  1. 移液器吸头1,000μL,200μL,10μL(Gilson,目录号:F1735001,F1733001,F1732001)。

  2. 1.7毫升PosiClick管(Denville Scientific,产品目录号:C2170)
  3. 2.0毫升安全锁管(Eppendorf,目录号:022363352)
  4. 质谱瓶和瓶盖Vex Kit,9 mm,PP,300μl+ PTFE /硅胶,预切口(Phenomenex,样本号:AR0-9992-13)。
  5. 不锈钢珠5毫米(QIAGEN,目录号:69989)
  6. 手套(Denville Scientific,目录编号:G4161)。
  7. 样品盘
  8. VanGuard墨盒架(WATERS,目录号:186007949)
  9. XBridge BEH Amide 2.5μmXP Vanguard Cartridge,2.1 x 5 mm(WATERS,产品目录号:186007763)。
  10. XBridge BEH Amide 2.5μm,2.1 x 100 mm色谱柱XP(WATERS,目录号:186006091)
  11. Acquity UPLC色谱柱在线过滤器套件(WATERS,产品目录号:205000343)
  12. MilliQ水-18mΩ,0.22μm过滤器(Merck,目录号:MPGP04001)
  13. 乙腈Optima LC / MS(Fisher Scientific,目录号:A955-4)。
  14. 醋酸铵(Sigma-Aldrich,目录号:73594-25G-F)。
  15. 氢氧化铵TraceMetal等级(Fisher Scientific,目录号:A512-P500)。
  16. 氩气压缩气体(Airgas)
  17. 氯仿(Fisher Scientific,目录号:C298-1)。
  18. 甲醇最佳LCMS(Fisher Scientific,目录号:A456-4)。
  19. 氮气(Airgas,目录号:NI230LT230RB)
  20. 溶剂A(见食谱)
  21. 溶剂B(见食谱)
  22. 密封洗(见食谱)

设备

  1. 移液器(P1000,P200,P20,P10)
  2. 离心机(Eppendorf,型号:5430 R,目录号:022620511)
  3. 涡流
  4. ACQUITY H-Class UPLC(沃特斯,型号:ACQUITY UPLC H-Class)
  5. Bath Sonicator(VWR,产品目录号:97043-976)
  6. 高速Vacuum-Vacufuge Plus(Eppendorf,型号:Vacufuge plus,产品目录号:022822993)
  7. TissueLyser II(QIAGEN,目录号:85300)
  8. Xevo TQ-S Micro(WATERS,型号:Xevo TQ-S Micro)

软件

  1. MassLynx版本4.1
  2. TargetLynx XS
  3. R(版本3.3)

程序

  1. 提取极性代谢物
    使用如下所述的改进的Bligh-Dyer方案提取极性代谢物(Bligh和Dyer,1959,Tambellini等人,2013)。除非另有说明,否则在冰上进行提取。
    1. 对于每个样品应该标记一组用于收集极性代谢物的1.5ml微量离心管。分析所需的生物学重复的次数取决于研究,对于高度控制的模型,其范围可以从少至5到数百到数千人类研究。
    2. 在开始提取方案之前,应准备以下物质并进行冷却:
      1. 2:1甲醇:氯仿混合物(每个样品300μl)。
      2. 纯甲醇(每个样品100μl)
      3. 水(18mΩ,0.22μm过滤)(每份100μl)
    3. 因此应该将离心机冷却到4℃。
    4. 应将样品置于冰上解冻。
      注意:
      1. 较小的样本大小可以成功使用。例如,在Krishnaiah等,2017中使用了1×10 6个U2OS细胞。
      2. 如果提取组织,应使用2ml安全锁定微量离心管。
    5. (可选)如果需要定量,则应在这些样品中添加以代谢物为基础确定浓度的稳定同位素标记内标。
    6. 一旦取样,如果提取细胞沉淀,按照步骤A6a或如果提取组织A6b。
      1. 细胞团块的提取:
        1. 向每个样品中加入300μl冷的2:1甲醇:氯仿混合物。
        2. 将每个微量离心管涡旋直至形成均匀的混合物。图1A显示了一个代表性的终点。
          注意:涡旋时细胞团粒可能不会被均质化,因此对样品进行超声处理是必不可少的。
        3. 接下来,将样品在35kHz下在浴超声波仪中超声处理15分钟。图1B显示了一个有代表性的终点。
        4. 超声处理结束后,将样品置于冰上。


          图1.提取过程中代表性终点的实例A.向其中加入300μl2:1甲醇:氯仿的细胞沉淀提取的实例并涡旋。 B.超声处理后细胞提取的一个例子(步骤A6a.iii)。 C.在离心结束时(步骤A10至A11)显示含水层(1)和有机层(2)。

      2. 使用TissueLyser II提取组织:

        1. 放置在-80°C预冷组织匀浆块
        2. 每个组织样本,小心添加300μl的冷2:1甲醇:氯仿混合物。
        3. 接下来,使用预冷块(使用手套的手柄),使用Tissue Lyser II将组织样品以25Hz匀化4分钟。
        4. 在均化结束时,将样品放回冰上。
    7. 接下来,将100μl的水和氯仿加入到每个含有样品(细胞团或组织)的微量离心管中。
    8. 然后将样品再次涡旋直至形成均匀的混合物。
    9. 为了分离极性和非极性代谢物,将包含样品的微量离心管在4℃以18.787xg(RCF)离心7分钟。
    10. 现在将样品小心地放在冰上,以避免分离层的破坏。
    11. 接下来,仔细收集含有极性代谢物的上层(在图1C中表示为层1)到预标记的微量离心管组中。 (如果需要,可以以类似的方式收集含有非极性代谢物的较低部分(在图1C中表示为层2)。)
      注意:建议在实际样品之前对测试样品进行提取程序。
    12. 然后将微量离心管在速度真空下干燥4小时或直至干燥。
    13. 在完成干燥后,代谢物可以如下所示重新悬浮或储存在-80℃
      注意:储存在-80°C的干燥样品不同化合物对代谢产物的影响不同,因此建议在提取后立即进行LC-MS分析。 >
  2. 重新悬浮并制备用于LC-MS的极性代谢物
    1. 样品制备顺序应该产生一个随机的顺序。
    2. 在分析开始时,除了10个平衡(EQ)样品以外,还将使用最少5个空白(参见本节步骤B13,了解有关准备EQ和QC样品的信息)。接下来,样品将进行4个质量控制(QC)样品。分析结束时,每6至10个样品,一个QC想要添加到2个QC(图2)。请注意QC注射之间的间距可能会有所不同,可能取决于样品数量。更好的质量控制频率(例如,每6个样品)提供更强大的数据校正,但需要更多样品。


      图2.显示样品运行的示例。应在分析开始时运行至少5个空白(蓝色),然后是10个平衡样品(绿色)。接下来是4个质量控制样品(红色),待分析样品(紫色),然后是2个质量控制样品。
      在整个分析过程中,6个样本可以从6到10个样本变化

    3. 1:1乙腈:水组成的稀释剂应事先准备和冷却
    4. 另外,离心机应该冷却到4℃。

    5. 干燥的极性代谢产物,稀释剂以及样品盘应置于冰上
    6. 含有干燥的极性代谢物的微量离心管以及空白的微量离心管。同样,将代谢物的体积重新悬浮在样品范围内并进行调整以确保它们被代谢。适当稀释代谢物可确保更长的柱寿命。
    7. 然后将每个离心管涡旋至少20秒,以重新悬浮代谢物。
    8. 接着,在离心机的18.787微量离心管7分钟×g的(RCF)在4℃下作为额外的预防步骤来清理样品。
    9. 然后如下将各样品的上清液等分:
      1. 5微升被添加到一个微离心管,以产生一个汇集的质量控制(QC)样品。例如,如果要运行10个QC样品,则如果要添加20μL,则将需要至少200μL到每个小瓶。)
      2. LC-MS样品瓶中至少添加20μl,用于分析每个重复样品。 (两种分析重复的最小,理想地3建议。)体积加入到每个小瓶中取决于注射体积为注射量的范围可以从2至10微升。 (已经注意到较小的注射量可以产生更好的色谱。)
        注意:建议您使用1.5 ml HPLC瓶。
    10. 根据预先随机的运行顺序将LC-MS样品瓶置于样品盘中。
    11. 将90μl的空白(或足够的空白数量)加入到LC-MS小瓶中并放入样品盘中。
    12. 一旦所有的样品都被分装,合并的QC样品被涡旋以确保均一性。
    13. 从汇集的QC样品:
      1. 通过用稀释剂(1:1乙腈:水)将QC样品稀释10倍来制备平衡样品。
      2. 将20μl添加到质量规格的样品瓶中,并将样品瓶放在样品盘中的适当位置。 (小瓶的数目取决于总QC样品上进行运行,并加入到小瓶中的体积变化的基础期望的注射体积。)
    14. 将90μl的EQ样品加入到LC-MS小瓶中并放入样品盘中。音量可以根据EQ的数量进行调整。

  3. 通过UPLC-MS获取数据
    然后使用通过COLMeD方法优化的方法(Rhoades and Weljie,2016)通过UPLC-MS分析极性代谢物。以下程序应作为获取质谱仪培训人员数据的指导原则。
    1. 溶剂A是根据配方1新鲜制备的。另外,存在足够的溶剂B(配方2)和密封剂洗涤剂(配方3)。如果不准备溶剂。
    2. 在XBRIDGE BEH酰胺柱(2.5微米100×2.1毫米)柱前在线过滤器和先锋在40℃的温度下,利用色谱柱用于与Waters Xevo三重四极杆质谱联用的Acquity(H-Class)UPLC系统。
    3. 样品管理器的温度设定为8°C。
    4. 另外,在分析过程中,氮气压力应保持在100PSI以上。
      使用氩气作为碰撞气体
    5. 在分析开始之前,溶剂管线,密封清洗和样品管理器都已准备就绪。另外,使用100%乙腈洗涤针头。
      在开始分析之前,质谱仪至少要运行30分钟
    6. “色谱柱维护和使用手册”概述如下。
    7. 表1用于每次进样的LC进样方法。每次注射的运行时间是30分钟,然后是1分钟的密封洗涤。

      表1. LC参数
      “”

    8. 在MSMS模式中用于质谱仪的参数在表2中示出,并且用于电喷雾正离子模式和负离子模式。

      表2.正面和负面模式的MS参数
      “”

    9. 注入2μl的空白和平衡样品,而2-10μl的进样体积用于质量对照样品和样品。
    10. 采用多反应监测(MRMs)处理样品,其中338种代谢物以其质荷比为目标。 MRM产生除了沃特世的IntelliStart软件和标准利用数据库METLIN(史密斯等人,2005年)和HMDB(威沙特等人,2013),以及(Basu和Blair,2011,Yuan等人,2012)。扫描代谢物及其设置(过渡和锥体和碰撞电压)中的表S1

数据分析

  1. 使用TargetLynx软件如上所述通过LC-MS采集的数据。总强度色谱图,整合它们并获得离子计数(图3)。

    “”
    图3.显示总强度色谱图的示例。在这里,甲硫氨酸的高峰被识别并使用TargetLynx进行整合,导致离子计数为994753.69。

  2. 目标TargetLynx现在被导出到一个文本文件,在R进一步处理。
  3. 在R中,定制的脚本用于去除QC样品少于50%的代谢物。该QC样品,以便用于拟合一个交叉验证局部估计散点图平滑函数(LOESS)每一代谢物(唐恩等人,2011),以便在的过程中考虑到仪器漂移分析用于离子计数的标准化(图4)。 Waters的TargetLynx软件。)
  4. 标准化离子计数,然后用于进一步的统计和多元分析。

    “”
    图4.显示的样本数据集的主成分分析(PCA) 平均两个分析重复的两个果蝇基因型(富民和不眠)的PCA示出质量控制(QC)样品的在数据分析过程中的各个步骤的群集。 LESS校正(B)QC样品(红色)在LOESS校正和归一化(A)之前显示。
    在LOESS校正数据(C)的中位倍数变化归一化之后,QC样品保持紧密聚集

笔记

上述提取方案已经在细胞沉淀的分析被利用(Krishnaiah 等人,2017),肝(Krishnaiah 等人,2017年),全果蝇( 。马奎尔等人,2015年),和脑组织(麦格拉思等人,2008)以及其它样品类型:诸如等离子体(Banoei 等。 ,2017),血清(丽怡等人,2016和豪等人,2016),cereberal脊髓液生物流体,酵母(Tambellini ,2013和2017)等我们也强烈建议没有任何结果通过包含提取或类似的矩阵,以确认的保留时间和裂解谱分析未标记的标准之前的稳定同位素标记的内标物进行验证。

食谱

  1. 溶剂A
    95:5水:乙腈
    20 mM醋酸铵
    氢氧化铵,pH值为9
    在35 kHz超声15分钟
  2. 溶剂B
    100%乙腈
    超声5分钟。
  3. 密封清洗
    50:50水:乙腈
    在35 kHz超声15分钟

确认

D.M.M和S.D.R.通过药理T32培训资助(T32 GM008076)并通过奖励AMW部分支持研究所转化医学和治疗学(ITMAT)跨学科项目在转化医学和宾夕法尼亚大学的治疗项目的支持。项目描述由国家研究资源中心,格兰特UL1RR024134什么支持,现在是国家中心为推进转化科学,格兰特UL1TR000003。内容完全是作者的责任,并不代表NIH的官方观点。上述协议已经从(罗兹和Weljie,2016)并适于(Krishnaiah 等人,2017年)。作者声明不存在利益冲突或利益冲突。

参考

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Malik, D. M., Rhoades, S. D. and Weljie, A. (2018). Extraction and Analysis of Pan-metabolome Polar Metabolites by Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS). Bio-protocol 8(3): e2715. DOI: 10.21769/BioProtoc.2715.
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