Premstaller A, Oberacher H, Walcher W, Timperio A M, Zolla L, Chervet J P, Cavusoglu N, van Dorsselaer A, Huber C G
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innsbruck, Austria.
Anal Chem. 2001 Jun 1;73(11):2390-6. doi: 10.1021/ac010046q.
The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.
使用四氢呋喃/癸醇作为致孔剂制备微孔聚（苯乙烯/二乙烯基苯）整体柱，能够通过反相高效液相色谱（RP-HPLC）快速且高效地分离肽和蛋白质。与传统的颗粒状多孔固定相不同，在传统固定相中，负载量是分子量的函数，对于60×0.2-mm的毛细管柱，整体柱对小肽和大蛋白质的负载量均在0.4 - 0.9 pmol范围内。使用3-nl毛细管检测池在214 nm处测量紫外吸光度得到的检测下限，对于八肽为500 amol，对于核糖核酸酶A为200 amol。当洗脱液中三氟乙酸的浓度从常用的0.1 - 0.2%降低到0.05%时，分离系统成功地与电喷雾电离质谱（ESI-MS）联用，代价仅是分离效率略有降低。ESI-MS对蛋白质的检测限在低飞摩尔范围内。从重建离子色谱图中提取了高质量的质谱图，并由此推导出肽和蛋白质的质量，质量准确度为50 - 150 ppm。通过对牛过氧化氢酶和人转铁蛋白的胰蛋白酶肽段进行质量指纹分析以及对与高等植物光系统II天线复合物相关的膜蛋白进行分析，证明了整体柱技术在蛋白质组学中的适用性。
