有机溶剂基蛋白质沉淀，在质谱分析之前实现强大的蛋白质组纯化
Summary
本方案描述了在受控条件下的溶剂型蛋白质沉淀，以便在质谱分析之前稳定快速地回收和纯化蛋白质组样品。
Abstract
虽然质谱（MS）仪器的多项进步改进了蛋白质组的定性和定量分析，但在MS之前分离，富集和处理蛋白质的更可靠的前端方法对于成功的蛋白质组表征至关重要。低且不一致的蛋白质回收率和残留杂质（如表面活性剂）对MS分析有害。与其他样品制备策略相比，蛋白质沉淀通常被认为是不可靠，耗时且技术上具有挑战性的。通过采用最佳的蛋白质沉淀方案可以克服这些问题。对于丙酮沉淀，特定盐的组合、温度控制、溶剂组成和沉淀时间至关重要，而氯仿/甲醇/水沉淀的效率取决于适当的移液和样品瓶操作。或者，这些沉淀方案在一次性旋转墨盒中简化和半自动化。本研究阐述了以传统形式使用一次性两级过滤和提取滤芯的溶剂型蛋白质沉淀的预期结果。这包括通过自下而上的LC-MS / MS分析对蛋白质组学混合物进行详细表征。基于SDS的工作流程相对于未受污染的蛋白质也表现出卓越的性能。
Introduction
由于现代MS仪器的灵敏度，分辨率，扫描速度和多功能性的增强，质谱法的蛋白质组分析变得越来越严格。MS的进步有助于提高蛋白质鉴定效率和更精确的定量1，2，3，4，5。通过改进的MS仪器，研究人员需要一种相应一致的前端样品制备策略，能够在工作流程的所有阶段在最短的时间内定量回收高纯度蛋白质6，7，8，9，10，11.为了准确反映生物系统的蛋白质组状态，必须以高效和无偏倚的方式从天然样品基质中分离蛋白质。为此，包括变性表面活性剂，如十二烷基硫酸钠（SDS），确保蛋白质12的高效提取和增溶。然而，SDS强烈干扰电喷雾电离，如果不能适当地消除13，则引起严重的MS信号抑制。
各种SDS耗尽策略可用于随后的蛋白质组分析，例如保留高于一次性旋转柱14，15，16中包含的分子量截止过滤器的蛋白质。过滤辅助样品制备方法（FASP）受到青睐，因为它有效地将SDS消耗在10 ppm以下，从而促进最佳MS。然而，FASP的蛋白质回收率是可变的，这促使了对其他技术的探索。选择性捕获蛋白质（或表面活性剂）的色谱方法已经演变成各种方便的墨盒或基于磁珠的格式17，18，19，20，21。鉴于这些简单且（理想情况下）一致的蛋白质纯化策略，使用有机溶剂进行蛋白质沉淀的经典方法经常被忽视为一种有前途的蛋白质分离方法。虽然溶剂沉淀被证明可以成功地将SDS消耗到临界水平以下，但蛋白质回收一直是这种方法的长期关注点。多个研究小组观察到蛋白质回收偏倚，沉淀率与蛋白质浓度、分子量和疏水性22，23的函数关系低得令人无法接受。由于文献中报道的降水方案的多样性，开发了标准化的降水条件。2013年，Crowell等人首次报道了离子强度对80%丙酮24中蛋白质沉淀效率的依赖性。对于检查的所有蛋白质，添加高达30 mM的氯化钠对于最大化产量（高达100%回收率）至关重要。最近，Nickerson等人表明，在丙酮沉淀期间，更高的离子强度（高达100mM）与高温（20°C）的组合在2-5 min25内给出了近乎定量的回收。观察到低分子量（LMW）蛋白的回收率略有下降。因此，Baghalabadi等人的后续报告证明，通过将特定盐，特别是硫酸锌与更高水平的有机溶剂（97%丙酮）26结合，可以成功回收LMW蛋白和肽（≤5 kDa）26。
虽然改进沉淀方案为基于MS的蛋白质组学提供了更可靠的蛋白质纯化策略，但常规沉淀的成功在很大程度上取决于用户技术。这项工作的主要目标是提出一种强大的沉淀策略，有助于从污染的上清液中分离蛋白质沉淀。开发了一种一次性过滤滤芯，通过在多孔PTFE膜过滤器27上方分离聚集的蛋白质来消除移液。上清液中的MS干扰组分在短时间的低速离心步骤中被有效去除。一次性滤芯还提供可互换的 SPE 滤芯，便于在质谱分析之前进行再溶解和可选蛋白质消解后的后续样品清理。
这里介绍了一系列推荐的蛋白质组沉淀工作流程，包括常规（基于小瓶）和半自动格式的常规（基于小瓶）的改性丙酮和氯仿/甲醇/水28 方案，以及一次性双态过滤和提取滤芯中的半自动形式。突出显示了所得的蛋白质回收率和SDS消耗效率，以及自下而上的LC-MS / MS蛋白质组覆盖率，以证明每个方案的预期结果。讨论了与每种方法相关的实际优点和缺点。
Protocol
1. 材料考虑和样品预制备 仅使用高纯度溶剂（丙酮，氯仿，甲醇）（>99.5%）和化学品，不含过量水分。 在水中制备氯化钠和硫酸锌溶液（1M）。注意：盐溶液可以在室温下无限期储存，只要它们没有污染物或微生物生长。 使用最小的聚丙烯（PP）微量离心机小瓶，足以保留所需体积的样品和溶剂以诱导沉淀。 确保待沉淀样品中的SDS浓度不大于2%（w / v…
Representative Results
图4 总结了使用丙酮在一次性滤芯中基于小瓶或盒式促进的蛋白质沉淀后预期的SDS耗尽。将常规的丙酮过夜孵育（-20°C）与室温下的快速丙酮沉淀方案（步骤2）以及CMW沉淀（步骤4）进行比较。残余SDS通过亚甲基蓝活性物质（MBAS）测定29进行定量。简而言之，将100μL样品与100μLMBAS试剂（250mg亚甲基蓝，50g硫酸钠，10mL硫酸，在水中稀释至1.0L）混合，然后加入4…
Discussion
当残余SDS耗尽到10 ppm以下时，可实现最佳的MS表征。虽然FASP和磁珠消化等替代方法提供定量SDS耗尽，回收率为31，32，33，但沉淀的主要目标是同时最大化纯度和产量。这取决于在不干扰蛋白质沉淀的情况下有效分离上清液（含有SDS）。使用基于小瓶的沉淀，一旦通过移液去除了大部分上清液，一些聚集的沉淀物就越来越有可能…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作由加拿大自然科学和工程研究委员会资助。作者感谢生物信息学解决方案公司（加拿大滑铁卢）和病童医院（加拿大多伦多）的SPARC BioCentre（分子分析）对获取MS数据的贡献。
Materials
| Acetone | Fisher Scientific | AC177170010 | ≤0.002 % aldehyde |
| Acetonitrile | Fisher Scientific | A998-4 | HPLC grade |
| Ammonium Bicarbonate | Millipore Sigma | A6141-1KG | solid |
| Beta mercaptoethanol | Millipore Sigma | M3148-25ML | Molecular biology grade |
| Bromophenol blue | Millipore Sigma | B8026-5G | Bromophenol blue sodium salt |
| Chloroform | Fisher Scientific | C298-400 | Chloroform |
| Formic Acid | Honeywell | 56302 | Eluent additive for LC-MS |
| Fusion Lumos Mass Spectrometer | ThermoFisher Scientific | for analysis of standard protein mixture | |
| Glycerol | Millipore Sigma | 356352-1L-M | For molecular biology, > 99% |
| Isopropanol | Fisher Scientific | A4641 | HPLC grade |
| Methanol | Fisher Scientific | A452SK-4 | HPLC grade |
| Microcentrifuge | Fisher Scientific | 75-400-102 | up to 21,000 xg |
| Microcentrifuge Tube (1.5 mL) | Fisher Scientific | 05-408-130 | tapered bottom |
| Microcentrifuge Tube (2 mL) | Fisher Scientific | 02-681-321 | rounded bottom |
| Micropipette Tips (0.1-10 μL) | Fisher Scientific | 21-197-28 | Universal pipet tip, non-sterile |
| Micropipette Tips (1-200 μL) | Fisher Scientific | 07-200-302 | Universal pipet tip, non-sterile |
| Micropipette Tips (200-1000 μL) | Fisher Scientific | 07-200-303 | Universal pipet tip, non-sterile |
| Micropipettes | Fisher Scientific | 13-710-903 | Micropipet Trio pack |
| Pepsin | Millipore Sigma | P0525000 | Lyophilized powder, >3200 units/ mg |
| ProTrap XG | Proteoform Scientific | PXG-0002 | 50 complete units per box |
| Sodium Chloride | Millipore Sigma | S9888-1KG | ACS reagent, >99 % |
| Sodium Dodecyl Sulfate | ThermoFisher Scientific | 28312 | powdered solid |
| timsTOF Pro Mass Spectrometer | Bruker | for analysis of liver proteome extract | |
| Trifluoroacetic Acid | ThermoFisher Scientific | L06374.AP | 99% |
| Tris | Fisher Scientific | BP152-500 | Molecular biology grade |
| Trypsin | Millipore Sigma | 9002-07-7 | From bovine pancreas, TPCK-treated |
| Urea | Bio-Rad | 1610731 | solid |
| Water (deionized) | Sartorius Arium Mini Water Purification System | 76307-662 | Type 1 ultrapure (18.2 MΩ cm) |
| Zinc Sulfate | Millipore Sigma | 307491-100G | solid |
Referencias
- Kilpatrick, L. E., Kilpatrick, E. L. Optimizing high-resolution mass spectrometry for the identification of low-abundance post-translational modifications of intact proteins. Journal of Proteome Research. 16 (9), 3255-3265 (2017).
- Scheffler, K., Viner, R., Damoc, E. High resolution top-down experimental strategies on the Orbitrap platform. Journal of Proteomics. 175, 42-55 (2018).
- Quaranta, A., et al. N -Glycosylation profiling of intact target proteins by high-resolution mass spectrometry (MS) and glycan analysis using ion mobility-MS/MS. Analyst. 145 (5), 1737-1748 (2020).
- Van Der Burgt, Y. E. M., et al. Structural analysis of monoclonal antibodies by ultrahigh resolution MALDI in-source decay FT-ICR mass spectrometry. Analytical Chemistry. 91 (3), 2079-2085 (2019).
- Anderson, L. C., et al. Identification and characterization of human proteoforms by top-down LC-21 Tesla FT-ICR mass spectrometry. Journal of Proteome Research. 16 (2), 1087-1096 (2017).
- Nickerson, J. L., et al. Recent advances in top-down proteome sample processing ahead of MS analysis. Mass Spectrometry Reviews. , (2021).
- Kelly, R. T. Single-cell Proteomics: Progress and Prospects. Molecular and Cellular Proteomics. 19 (11), 1739-1748 (2020).
- Alexovič, M., Sabo, J., Longuespée, R. Microproteomic sample preparation. Proteomics. 21 (9), 2000318 (2021).
- Shishkova, E., Coon, J. J. Rapid preparation of human blood plasma for bottom-up proteomics analysis. STAR Protocols. 2 (4), 100856 (2021).
- Duong, V. A., Park, J. M., Lee, H. Review of three-dimensional liquid chromatography platforms for bottom-up proteomics. International Journal of Molecular Sciences. 21 (4), 1524 (2020).
- Gan, G., et al. SCASP: A simple and robust SDS-aided sample preparation method for proteomic research. Molecular and Cellular Proteomics. 20, 100051 (2021).
- Kachuk, C., Doucette, A. A. The benefits (and misfortunes) of SDS in top-down proteomics. Journal of Proteomics. 175, 75-86 (2018).
- Rundlett, K. L., Armstrong, D. W. Mechanism of signal suppression by anionic surfactants in capillary electrophoresis-electrospray ionization mass spectrometry. Analytical Chemistry. 68 (19), 3493-3497 (1996).
- Wis, J. R., Zougman, A., Nagaraj, N., Mann, M. Universal sample preparation method for proteome analysis. Nature Methods. 6 (5), 359-362 (2009).
- Ni, M., et al. Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics. Rapid Communications in Mass Spectrometry. 31 (2), 171-178 (2017).
- Zhao, Q., et al. imFASP: An integrated approach combining in-situ filter-aided sample pretreatment with microwave-assisted protein digestion for fast and efficient proteome sample preparation. Analytica Chimica Acta. 912, 58-64 (2016).
- Kanshin, E., et al. Ultrasensitive proteome analysis using paramagnetic bead technology. Molecular Systems Biology. 10 (10), 757 (2014).
- Hughes, C. S., et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nature Protocols. 14 (1), 68-85 (2019).
- Dagley, L. F., Infusini, G., Larsen, R. H., Sandow, J. J., Webb, A. I. Universal solid-phase protein preparation (USP3) for bottom-up and top-down proteomics. Journal of Proteome Research. 18 (7), 2915-2924 (2019).
- Hengel, S. M., et al. Evaluation of SDS depletion using an affinity spin column and IMS-MS detection. Proteomics. 12 (21), 3138-3142 (2012).
- Zougman, A., Selby, P. J., Banks, R. E. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. PROTEOMICS. 14 (9), 1006-1010 (2014).
- Thongboonkerd, V., McLeish, K. R., Arthur, J. M., Klein, J. B. Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation. Kidney International. 62 (4), 1461-1469 (2002).
- Klont, F., et al. Assessment of sample preparation bias in mass spectrometry-based proteomics. Analytical Chemistry. 90 (8), 5405-5413 (2018).
- Crowell, A. M. J., Wall, M. J., Doucette, A. A. Maximizing recovery of water-soluble proteins through acetone precipitation. Analytica Chimica Acta. 796, 48-54 (2013).
- Nickerson, J. L., Doucette, A. A. Rapid and quantitative protein precipitation for proteome analysis by mass spectrometry. Journal of Proteome Research. 19 (5), 2035-2042 (2020).
- Baghalabadi, V., Doucette, A. A. Mass spectrometry profiling of low molecular weight proteins and peptides isolated by acetone precipitation. Analytica Chimica Acta. 1138, 38-48 (2020).
- Crowell, A. M. J., MacLellan, D. L., Doucette, A. A. A two-stage spin cartridge for integrated protein precipitation, digestion and SDS removal in a comparative bottom-up proteomics workflow. Journal of Proteomics. 118, 140-150 (2015).
- Wessel, D., Flügge, U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry. 138 (1), 141-143 (1984).
- Arand, M., Friedberg, T., Oesch, F. Colorimetric quantitation of trace amounts of sodium lauryl sulfate in the presence of nucleic acids and proteins. Analytical Biochemistry. 207 (1), 73-75 (1992).
- Tukey, J. W. Exploratory data analysis. Biometrics. 33, 768 (1977).
- Ludwig, K. R., Schroll, M. M., Hummon, A. B. Comparison of in-solution, FASP, and S-Trap based digestion methods for bottom-up proteomic studies. Journal of Proteome Research. 17 (7), 2480-2490 (2018).
- Sielaff, M., et al. Evaluation of FASP, SP3, and iST protocols for proteomic sample preparation in the low microgram range. Journal of Proteome Research. 16 (11), 4060-4072 (2017).
- Supasri, K. M., et al. Evaluation of filter, paramagnetic, and STAGETips aided workflows for proteome profiling of symbiodiniaceae. Processes. 9 (6), 983 (2021).
- Botelho, D., et al. Top-down and bottom-up proteomics of SDS-containing solutions following mass-based separation. Journal of Proteome Research. 9 (6), 2863-2870 (2010).
- Fic, E., Kedracka-Krok, S., Jankowska, U., Pirog, A., Dziedzicka-Wasylewska, M. Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis. Electrophoresis. 31 (21), 3573-3579 (2010).
- Antonioli, P., Bachi, A., Fasoli, E., Righetti, P. G. Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis. Journal of Chromatography A. 1216 (17), 3606-3612 (2009).
- Bryzgunova, O., et al. A reliable method to concentrate circulating DNA. Analytical Biochemistry. 408 (2), 354-356 (2011).
- Asakura, T., Adachi, K., Schwartz, E. Stabilizing effect of various organic solvents on protein. Journal of Biological Chemistry. 253 (18), 6423-6425 (1978).
- Loo, J. A., Loo, R. R. O., Udseth, H. R., Edmonds, C. G., Smith, R. D. Solvent-induced conformational changes of polypeptides probed by electrospray-ionization mass spectrometry. Rapid Communications in Mass Spectrometry. 5 (3), 101-105 (1991).
- Simpson, D. M., Beynon, R. J. Acetone precipitation of proteins and the modification of peptides. Journal of Proteome Research. 9 (1), 444-450 (2010).
- Güray, M. Z., Zheng, S., Doucette, A. A. Mass spectrometry of intact proteins reveals +98 u chemical artifacts following precipitation in acetone. Journal of Proteome Research. 16 (2), 889-897 (2017).
- Doucette, A. A., Vieira, D. B., Orton, D. J., Wall, M. J. Resolubilization of precipitated intact membrane proteins with cold formic acid for analysis by mass spectrometry. Journal of Proteome Research. 13 (12), 6001-6012 (2014).
- Beavis, R. C., Chait, B. T. Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America. 87 (17), 6873-6877 (1992).
- Klunk, W. E., Pettegrew, J. W. Alzheimer’s β-Amyloid protein is covalently modified when dissolved in formic acid. Journal of Neurochemistry. 54 (6), 2050-2056 (1990).
- Vuckovic, D., Dagley, L. F., Purcell, A. W., Emili, A. Membrane proteomics by high performance liquid chromatography-tandem mass spectrometry: Analytical approaches and challenges. Proteomics. 13 (3-4), 404-423 (2013).
- Smith, L. M., et al. The human proteoform project: Defining the human proteome. Science Advances. 7 (46), (2021).
Citar este artículo
Nickerson, J. L., Baghalabadi, V., Dang, Z., Miller, V. A., Little, S. L., Doucette, A. A. Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry. J. Vis. Exp. (180), e63503, doi:10.3791/63503 (2022).