Schley Christian, Swart Remco, Huber Christian G
Department of Chemistry, Instrumental Analysis and Bioanalysis, Saarland University, 66123 Saarbrücken, Germany.
J Chromatogr A. 2006 Dec 15;1136(2):210-20. doi: 10.1016/j.chroma.2006.09.072. Epub 2006 Oct 17.
Monolithic columns based on poly-(styrene-divinylbenzene) (PS-DVB) were utilized both for preconcentration (in 10 mm x 0.20 mm I.D. format) and analytical separation (in 60 mm x 0.20 and 0.10 mm I.D. format) of peptides and proteins in column switching micro-scale high-performance liquid chromatography. A special holder for short monolithic preconcentration columns was designed and pressure durability tests approved long-term stability up to 400 bar. An 11-20% decrease in the average peak widths of nine peptides was obtained upon combining a preconcentration column with an analytical column as compared with a setup using an analytical column only. Trapping efficiency, especially for small and hydrophilic peptides, was optimized by using 0.10% heptafluorobutyric acid instead of 0.050% trifluoroacetic acid as solvent additive during sample loading. Using a 10 mm x 0.20 mm I.D. preconcentration column, loadabilities between 0.5 and 1.6 microg were determined by frontal analysis of proteins and bioactive peptides, respectively. A 100-fold concentration followed by direct on-line intact mass determination is demonstrated for diluted (3 micromolL(-1)) protein solutions. The applicability of the monolithic preconcentration column for multidimensional chromatography was tested by off-line two-dimensional separation, combining strong cation-exchange chromatography and ion-pair reversed-phase chromatography. Peptide identification data from digested protein mixtures demonstrated reproducibilities of 46-75% in triplicate analyses, and confident peptide identifications of low abundant peptides even in the presence of a 650-fold molar excess of high abundant peptides.
基于聚（苯乙烯 - 二乙烯基苯）（PS - DVB）的整体柱被用于柱切换微尺度高效液相色谱中肽和蛋白质的预浓缩（10 mm×0.20 mm内径规格）和分析分离（60 mm×0.20和0.10 mm内径规格）。设计了一种用于短整体预浓缩柱的特殊固定器，压力耐久性测试证明其在高达400 bar的压力下具有长期稳定性。与仅使用分析柱的设置相比，将预浓缩柱与分析柱结合使用时，九种肽的平均峰宽降低了11 - 20%。在进样期间，通过使用0.10%的七氟丁酸代替0.050%的三氟乙酸作为溶剂添加剂，优化了捕集效率，尤其是对于小的亲水性肽。使用10 mm×0.20 mm内径的预浓缩柱，通过蛋白质和生物活性肽的前沿分析分别测定了0.5至1.6 μg的负载量。对于稀释的（3 μmolL⁻¹）蛋白质溶液，展示了100倍浓缩后直接在线进行完整质量测定的过程。通过离线二维分离，结合强阳离子交换色谱和离子对反相色谱，测试了整体预浓缩柱在多维色谱中的适用性。来自消化蛋白质混合物的肽鉴定数据在三次重复分析中显示出46 - 75%的重现性，并且即使在存在650倍摩尔过量的高丰度肽的情况下，也能可靠地鉴定低丰度肽。
