National Institute of Chemistry, Laboratory for Food Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.
J Chromatogr A. 2012 Mar 2;1227:210-8. doi: 10.1016/j.chroma.2012.01.011. Epub 2012 Jan 10.
The separation and isolation of major whey proteins is already extensively covered in the literature although no study has been published in which monolithic columns were used. In our research we present, for the first time, the use of short convective interaction media (CIM) monolithic columns for the separation of all major whey proteins and isolation of β-lactoglobulin variant A and B (β-LgA and β-LgB) from a commercial product whey isolate (WI). Although our primary interest was directed towards finding a proper monolithic column and chromatographic conditions for the purification and isolation of β-LgA and β-LgB, three additional analytical LC methods, each having its own potential application target, were also developed in the course of our research. On the monolithic diethylaminoethyl convective interaction media analytical column (CIMac DEAE), the separation of major whey proteins was achieved by gradually lowering the pH of the mobile phase. The ever-so-hard obtainable linear external pH gradient was very linear in the range of pH 5.5-3 and the developed ion-exchange (IE) high-performance liquid chromatographic (HPLC) method was amenable to mass spectrometry (MS). A very fast baseline separation, with UV detection, of all major whey proteins was achieved on a prototype CIMac reversed-phase styrene-divinylbenzene (RP-SDVB) monolithic column in only 4 min and the performance of this column proved superior in comparison with the packed particle POROS perfusion column. The developed RP-HPLC-MS method is fast and, due to the MS detector, can offer low limits of detection and quantitation. Finally, in order to fulfill our primary interest, a scale-up method was developed, using a prototype 8 mL analogue of the CIMac RP-SDVB column, for the isolation of native and chemically unmodified β-LgA and β-LgB from WI with purities higher than 90% and 81%, respectively. The proteins were to be used in further protein-ligand binding studies. The developed methods excel in speed of the analysis, sensitivity, resolution, and simplicity. Thus, it is shown for the first time that short monolithic columns are applicable to the separation and isolation of major whey proteins and that their use has some obvious benefits.
乳清蛋白的分离和纯化在文献中已有广泛的报道，但尚未有研究使用整体柱进行乳清蛋白的分离。在本研究中，我们首次提出使用短的整体柱作为分离乳清中主要蛋白和分离β-乳球蛋白变体 A 和 B（β-LgA 和 β-LgB）的手段，使用的整体柱是短的二乙基氨基乙基整体柱（CIMac DEAE）。虽然我们的主要兴趣是寻找合适的整体柱和色谱条件来分离和纯化β-LgA 和β-LgB，但在研究过程中还开发了另外三种具有各自潜在应用目标的分析型 LC 方法。在 CIMac 二乙基氨基乙基整体柱上，通过逐渐降低流动相的 pH 值来实现乳清中主要蛋白的分离。通过外部 pH 值线性梯度洗脱，可以获得非常线性的分离效果，pH 值范围为 5.5-3。开发的离子交换（IE）高效液相色谱（HPLC）方法适合与质谱（MS）联用。在原型 CIMac 反相苯乙烯-二乙烯基苯（RP-SDVB）整体柱上，仅用 4 分钟即可实现所有主要乳清蛋白的快速基线分离（UV 检测），并且该柱的性能优于填充颗粒 POROS 灌注柱。所开发的 RP-HPLC-MS 方法快速，并且由于 MS 检测器的存在，该方法可以提供较低的检测限和定量限。最后，为了满足我们的主要研究兴趣，我们使用原型 8 mL 模拟 CIMac RP-SDVB 整体柱开发了放大方法，从 WI 中分离天然和未经化学修饰的β-LgA 和β-LgB，纯度分别高于 90%和 81%。这些蛋白质将用于进一步的蛋白质-配体结合研究。所开发的方法在分析速度、灵敏度、分辨率和简单性方面表现出色。因此，首次证明短的整体柱适用于乳清蛋白的分离和纯化，并且它们的使用具有一些明显的优势。
