一个定制HPLC纯化协议，产量高纯度淀粉样蛋白β42和贝塔淀粉样蛋白肽40，有能力寡聚物形成的
Summary
在此，我们报告说，得到高纯度的β淀粉样蛋白42（Aβ42）和β淀粉样蛋白40（Aβ40）肽，能够形成低聚物量身定制的HPLC纯化协议。 β淀粉样蛋白在阿尔茨海默氏症牵连高度易聚集，疏水肽。该肽的淀粉样蛋白性质使得其净化是一个挑战。
Abstract
Amyloidogenic peptides such as the Alzheimer’s disease-implicated Amyloid beta (Aβ), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase – styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (>95%) Aβ42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the Aβ40 peptide with identical success and without the need to alter the method.
Introduction
阿尔茨海默氏症是一种神经退行性疾病的全球影响超过35万人。 1在疾病的发作和发展强烈牵连，是高度易聚集，疏水肽β淀粉样蛋白（Aβ）。 2Aβ从长度36至43个氨基酸的范围内，但是，它被认为是42-氨基酸变体，淀粉样蛋白β42（Aβ42），是蛋白质的最毒的形式。 3这是由于在大多数情况下，以Aβ42的容易地形成被认为是特别神经毒性实体扩散，低聚物质的能力。 4为了进一步提供了对Aβ肽的了解，有必要定期地获得高纯度的材料。微量杂质的存在已经显示出显着改变肽的聚集倾向的属性。 五
ŧraditionally，高效液相色谱（HPLC）的疏水性肽，例如Aβ的分离已通过使用C 4或C 8的二氧化硅基固定相和酸性流动相的组合完成的。 6然而，这样的条件可以提出一个挑战的肽的纯化。所述Aβ肽的低等电点（pI约5.5）7意味着在酸性条件下，肽聚集增加，并且作为结果宽，非分辨的HPLC峰是常常难以分离产生（ 图2A）。此外，这样的宽峰常常含有可能影响所述肽的聚集的空间，通常需要后续的轮次的纯化，其可以极大地影响肽的产生量的杂质。
的聚（苯乙烯/二乙烯基苯）固定相，PLRP-S，表示PURIF的替代手段英疏水性肽。固定相已在许多不同的蛋白质和信使核糖核酸（mRNA）的的纯化被使用。 8，9 PLRP-S固定相需要用于反相分离没有额外的烷基配体，而且更重要的是化学稳定在高pH导致肽的解聚。 7在此，我们报告说，得到高纯度的β淀粉样蛋白42（Aβ42）和β淀粉样蛋白40（Aβ40）肽量身定制的HPLC纯化协议。
Protocol
Representative Results
Discussion
所述Aβ肽的HPLC纯化是高度依赖于两者在纯化中使用的固定相和选择，以洗脱所述肽的流动相的选择。肽和高倾向聚集低等电点呈现疏水蛋白质的分离（C4或加上酸性移动洗脱液C8固定相）挑战传统色谱条件下，与Aβ肽洗脱作为延长的宽，非分辨峰（ 图2A）。
为了避免这个问题，PLRP-S固定相，在高pH下是化学稳定的被发现是有效的Aβ肽（ 图2B和<stron…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者想感谢安捷伦为他们提供技术援助。凯特·马卡姆和拉斐尔·帕洛米诺都记在合成的初始帮助纯化的Aβ肽和Hsiau伟李博士在准备手稿图1感谢他的帮助。
Materials
| Agilent 1260 Infinity II quarternary pump | Agilent | G7111B | http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1260-infinity-ii-quaternary-pump |
| Agilent 1260 Infinity II Dual variable wavelength detector | Agilent | G7114A | http://www.agilent.com/en-us/products/liquid-chromatography/lc-detectors/1260-infinity-ii-variable-wavelength-detector |
| Agilent 1260 Infinity II Manual Injector fitted with 10 mL stainless steel sample loop | Agilent | 0101-1232 | http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector |
| Agilent 1260 Infinity II Manual Injector fitted with 20 µL stainless steel sample loop | Agilent | G1328C | http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector |
| Ring Stand Mounting Bracket | Agilent | 1400-3166 | |
| PEEK Tubing Blue (1/32" outer diameter х 0.010" internal diameter) | Thermo Scientific | 03-050-399 | |
| Agilent PLRP-S 300Å 8µm 25 х 300 mm column (Preparative) | Agilent | PL1212-6801 | http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features |
| Agilent PLRP-S 300Å 8µm 7.5 х 300 mm (Semi-Preparative) | Agilent | PL1112-6801 | http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features |
| Agilent PLRP-S 300Å 5 µm 4.6 x 250 mm (Analytical) | Agilent | PL1512-5501 | http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features |
| Aβ42 or Aβ40 peptide | Synthesized in-house using a CEM liberty automated peptide synthesizer. | ||
| Ammonium Hydroxide (NH4OH, 28% solution) | Fisher Scientific | A669-500 | |
| Acetonitrile | Fisher Scientific | A998-4 | |
| HPLC grade water | Fisher Scientific | W5-4 | |
| Falcon 50 ml conical centrifuge tube | Fisher Scientific | 14-954-49A | |
| Supelco PEEK Fitting One-piece fingertight, pkg of 5 ea | Sigma-Aldrich | Z227250 | |
| Normject 5cc sterile syringe | Fisher Scientific | 1481729 | |
| 16 Gauge SS Needle | Rheodyne | 3725-086 |
References
- Querfurth, H. W., LaFerla, F. M. Alzheimer’s Disease. N. Engl. J. Med. 362 (4), 329-344 (2010).
- McGowan, E., et al. Aβ42 Is Essential for Parenchymal and Vascular Amyloid Deposition in Mice. Neuron. 47 (2), 191-199 (2005).
- Gong, Y., et al. Alzheimer’s disease-affected brain: Presence of oligomeric Aβ ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc. Natl. Acad. Sci. USA. 100 (18), 10417-10422 (2003).
- Selkoe, D. J. Soluble Oligomers of the Amyloid β-Protein Impair Synaptic Plasticity and Behavior. Behav Brain Res. 192 (1), 106-113 (2008).
- Zagorski, M. G., Yang, J., Shao, H., Ma, K., Zeng, H., Hong, A. Methodological and Chemical Factors Affecting Amyloid β Peptide Amyloidogenicity. Methods Enzymol. 309, 189-204 (1999).
- Kim, W., Hecht, M. H. Mutations Enhance the Aggregation Propensity of the Alzheimer’s Aβ Peptide. J Mol Bio. 377 (2), 565-574 (2008).
- Fezoui, Y., et al. An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments. Amyloid. 7 (3), 166-178 (2000).
- Zhelev, N. Z., Barratt, M. J., Mahadevan, L. C. Use of reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene columns for the rapid separation and purification of acid-soluble nuclear proteins. J Chromatogr A. 763 (1-2), 65-70 (1997).
- Thess, A., et al. Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals. Mol Ther. 23 (9), 1456-1464 (2015).
- Warner, C. J. A., Dutta, S., Foley, A. R., Raskatov, J. A. Introduction of D-glutamate at a critical residue of Aβ42 stabilizes a pre-fibrillary aggregate with enhanced toxicity. Chem Eur J. 22 (34), 11967-11970 (2016).
- Thompson, J. A., Lim, T. K., Barrow, C. J. On-line High-performance Liquid Chromatography/Mass Spectrometric Investigation of Amyloid-β Peptide Variants Found in Alzheimer’s Disease. Rapid Commun. Mass Spectrom. 13 (23), 2348-2351 (1999).
- Layne, E. Spectrophotometric and turbidimetric methods for measuring proteins. Met. Enzymology. 3, 447-455 (1957).
- Ioannou, J. C., Donald, A. M., Tromp, R. H. Characterizing the secondary structure changes occurring in high density systems of BLG dissolved in aqueous pH 3 buffer. Food Hydro. 46, 216-225 (2015).
- Rahimi, F., Maiti, P., Bitan, G. Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides. J. Vis. Exp. (23), e1071 (2009).
- Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways. Proc. Natl. Acad. Sci. USA. 100 (1), 330-335 (2003).
