肽中纯化检测方案(制备液相色谱)

Chromatography Application Note AN115 v1.0 Reversed Phase FlashChromatography Purification ofPeptide-Peptoid Hybrids Everywhereyoulook" Chromatography Application Note AN115，Jan 15， 2021 Vera DAloisio' and Christopher R. Coxon?* Application Overview Peptide-peptoid hybrids or ‘peptomers'are oligomeric synthetic polymers， inwhich one or more amino acids is replaced with apeptoid submonomer. Thekey structural feature is the relocation of the amino acid side chain from theo-carbon to the adjacent nitrogen atom of the peptide bond (see scheme inFig. 1). Generally， these species exhibit physicochemical properties similarto the parent peptides. Purification of peptide-peptoid hybrids can be obtained by reversed phasechromatography with excellent recovery and resolution. In general， foroptimising the separation of a mixture of peptides and similar compounds，it is best to consider parameters， such as sample loading and stationaryphase particle size. In this application note， the separation of a peptomerfrom a peptide side-product using Teledyne ISCO's RediSep C18 reversedphase columns as the stationary phase will be shown. Peptide-peptoid hybrid Figure 1. Peptides and peptomers Experimental Section The separation of a mixture ofone peptomer and one close-running peptide (retention times 9.34 and 8.06 min，respectively over a 20 min gradient H2O/MeCN as shown in Fig.3) was achieved with both RediSep Rf C18 4.3 gand RediSep Rf C18 Gold5.5 g，using the Teledyne ISCO CombiFlashNextGen 300+. Table 1 shows the sequencesof the peptomer and its side-product， which appears as a deletion of the parent compound. Parameters kept constantin this study are listed in Table 2， while the parameters that have been modified are showed in Table 3. Table 1. Peptomer sequence and side-product sequence.The letter F highlighted in bold is the amino acidreplaced with its peptoid equivalent. Instrumentation Teledyne ISCO CombiFlash NextGen 300+ Wavelengths 214 nm (red) 254 nm (purple) Mobile phases Solvent A： Purified H，OSolvent B： Methanol Flow Rate 13 mL/min EquilibrationVolume 28.7 mL Gradient % Solvent B 1010010090 90 Minute Initial 5 7.6 8 8.8 Run Length 8.8 min， not including equilibration time Table 2. Fixed parameters used in Flashchromatography separation Run N. Column Amount loaded (liquid loading) 1 RediSep Rf C18 4.3 g 0.5mL (8mg/mL) 2 RediSep Rf C18 5.5 g GOLD 0.5 mL (8 mg/mL) 3 RediSep Rf C18 4.3 g 1mL (8mg/mL) 4 Redi Sep Rf C18 5.5 g GOLD 1 mL (8 mg/mL) Table 3. Parameters changed in different runs. Characterization of crude and purified peptomer was performed with a low resolution LCMS Waters TQD massspectrometer equipped with an Agilent ZORBAX SB-C18 Stable Bond Analytical column (5 um particle size， 4.6x150 mm) with a binary eluent system comprising MeCN/H2O (20 min gradient： from 90%H2O/10% MeCN to100% MeCN with 0.1 % formic acid) as mobile phases. Electrospray ionisation mass spectrometry was conducted inpositive ion mode (m /z range： 600-1900) usinga cone voltage of 50 V， desolvation temperature of 350°C and sourcetemperature of 100 °C. Results When a low amount of peptide-peptomer mixture(8 mg) was loaded on C18 reversed phase 4.3 g RediSep column(Run 1)， flash chromatography successfully separated the product using water/methanol as the mobile phase (Figure1). No ion pairing reagent (e.g.TFA) was required for the separation. Higher peak resolution was obtained when thesame amount of initial mixture (8 mg) was loaded on a C18 reversed phase 5.5 g RediSep Gold column (Figure 2，Run 2). Loading a higher amount of crude mixture (16 mg) incurred a loss of resolution when a normal RediSep C18 4.3 gwas used (Run 3). In contrast， the successful separation at this scale when using a RediSep Gold C18 5.5 g (Run 4)highlights the importance of the use of a column with lower particle size when the separation is scaled up. Thegold column stationary phase composition exhibits a patented spherical Flash media， which affords the benefits oftighter packing without increasing back pressure. The resolving power is doubled compared with a standard flashchromatography column， allowing the separation of difficult compounds with low ARf， such as a parent peptomerand truncation side-product and， at the same time， allowing the loading of twice as much compound without losingresolution of the peaks. Figure 2. Chromatograms from separations obtained by varying the sample amount and the column media. Figure 3. HPLC chromatograms before and after separation. Fraction 1 contains the side-productof the parent sequence， while Fraction 2 contains the desired purified peptomer. Conclusions The separation of polar compounds such as peptides and peptidomimetics is usually achieved by reversed phasepreparative-HPLC due .tueo t(the typically higher resolving power compared to traditional flash chromatography.Separating closely related peptides， differing by one or two amino acids， can be challenging even when usingreversed-phase liquid chromatography. The use of preparative-HPLC is also time-consuming，requires expensive columns，with the possibility of loading only small amounts of sample in each run， meaning that multiple injections arerequired. Flash chromatography using pre-packed C18 reversed-phase columns provides convenient and comparablepreparative separations with automated flash chromatography instrumentation. In this work， separation of a peptomer from its side product was successfully achieved using flash chromatographypre-packed columns. This allowed the introduction of higher crude sample amounts and achieved purity comparablewith preparative-HPLC but requiring a shorter run time (8.8 min). The peak resolution was clearly influenced by thecolumn particle size and the loading， suggesting that for higher loading and difficult separation， the extra resolutionof the RediSep Gold C18 technology is key for successful separation. ' School of Pharmacy and Biomolecular Sciences， Faculty of Science， Liverpool John Moores University，Liverpool， United Kingdom， L3 3AF 2*Corresponding author， Institute of Chemical Sciences， School of Engineering and Physical Sciences，Heriot-Watt University， Edinburgh， United Kingdom， EH14 4AS Teledyne ISCO P.O. Box 82531， Lincoln， Nebraska，68501 USA Toll-free：(800) 228-4373·Phone： (402) 464-0231·Fax： (402) 465-3091 Teledyne ISCO is continually improving its products and reserves the right to change productspecifications， replacement parts， schematics，and instructions without notice. 应用概述肽-类肽杂交体或“肽聚体”是寡聚合成的聚合物，其中一个或多个氨基酸被类肽亚单体取代。其关键结构特征是氨基酸的侧链从α-碳重排到肽键的相邻氮原子（参见图1）。通常，这类物质表现出与亲本肽相似的理化特性。肽-类肽杂交体的纯化可以通过具有出色回收率和分辨率的反相色谱法获得。通常，优化肽和类似化合物混合物的分离，最好是考虑调整参数，比如上样量和固定相的粒径。本应用将讲述，使用Teledyne ISCO的RediSep® C18反相色谱柱作为固定相，从肽副产物中分离出肽聚体。 图 1. 肽和类肽 实验部分使用 Teledyne ISCO CombiFlash® NextGen 300+，在RediSep®Rf C18 4.3g色谱柱和 RediSep Rf C18 Gold® 5.5 g色谱柱上，分别实现了一种肽聚体和一种相近的肽的混合物分离（如图3所示，在20分钟的H2O/MeCN梯度中，保留时间分别为9.34和8.06分钟）。表1给出了 肽聚体及其副产物的序列，其中副产物的分子结构比母体化合物少一个氨基酸。本研究中保持不变的参数在表2给出，变化的参数在表3中。用低分辨率LCMS Waters TQD质谱仪对粗制和纯化的肽聚体进行表征，色谱柱使用Agilent ZORBAX SB-C18 Stable Bond Analytical（5μm粒径，4.6x150mm），流动相为MeCN/H2O二元洗脱体系，20分钟梯度从90% H2O/10% MeCN到100% MeCN（含 0.1% 甲酸）。喷雾电离质谱在正离子模式（m/z范围：600–1900）运行，锥孔电压为50V，去化温度350℃，源温度为100℃。结果用少量肽-肽聚体混合物(8mg)加载到C18反相4.3g RediSep色谱柱（运行1），使用水/甲醇作为流动相快速且成功地分离了产物（图1）。此时分离不需要配对离子试剂（如TFA）。当使用C18反相5.5 g RediSep Gold 色谱柱时，对于相同的上样量(8mg)，可获得更高的峰分辨率（图 2，运行2）。当加载更大量的粗混合物(16 mg)时，使用常规RediSep C18 4.3g色谱柱（运行3），出现分辨率损失的情况。相比之下，使用RediSep Gold C18 5.5g色谱柱（运行4），在同规模下可成功实现分离。这就充分说明了，在扩大分离规模时使用较小粒径色谱柱的重要性。金标柱固定相组合物采用获得专利的球形Flash介质，可在不增加背压的情况下提供更紧密的填充。与标准快速色谱柱相比，分辨率提高了一倍，可以分离具有低ΔRf的困难化合物，例如母体肽聚物和截断副产物，同时，可以加载两倍的化合物，也不会使峰的分辨率损失。结论与传统快速色谱法相比，通常通过反相制备型HPLC实现极性化合物的分离，如肽和类肽模拟物，因为其分离能力通常更高。即使在使用反相液相色谱时，分离具有一或两个氨基酸差异的高度相似的肽也非常困难。制备型HPLC的使用也很耗时，需要昂贵的色谱柱，而且运行时每次的样品加载量可能也很有限，因而要多次进样。预装的C18反相柱为快速色谱提供使用便利，并且其制备分离与自动快速色谱仪具有可比性。在本工作中，使用快速色谱预装柱成功地实现了肽聚合物与其副产物的分离。在更短的运行时间（8.8 分钟）内，同时允许加载更高的粗样品量并获得与制备型HPLC相当的纯化结果。峰的分辨率明显受色谱柱粒径和负载量的影响，表明对于更高的负载量和较困难的分离，由RediSep Gold C18技术的超高度分辨率是分离成功的关键。