Pirmoradian Mohammad, Zhang Bo, Chingin Konstantin, Astorga-Wells Juan, Zubarev Roman A
Department of Medical Biochemistry and Biophysics, Karolinska Institutet , Scheelesväg 2, SE-17177 Stockholm, Sweden.
Anal Chem. 2014 Jun 17;86(12):5728-32. doi: 10.1021/ac404180e. Epub 2014 May 28.
Recently, we introduced an online multijunction capillary isoelectric focusing (OMJ-CIEF) fractionator to fractionate proteins and peptides in electrospray-friendly solution. In this follow-up study, the original configuration of the fractionator was modified to improve the resolving power and reproducibility of separation. The major improvements include stabilization of the electrical current through the device using a voltage divider and stepwise elution of peptide zones in conjunction with the repeated refocusing of remaining peptides. Also, a novel algorithm was developed to calculate more accurately the pI values of peptides identified from experimental data. The standard deviation of calculated pI values for unmodified peptides from the theoretically predicted pI values was on average 0.21 pH units, which is more accurate than in standard-resolution gel-based methods. In order to characterize the analytical performance of the improved device, it was applied for the pI fractionation of yeast proteome digest into 18 fractions, with the collected fractions being analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry. Approximately 37% of 20047 identified peptides were detected in only one fraction and 27% - in two fractions. On average, every peptide was found in 2.4 fractions. These results strongly indicate the suitability of the improved device as a first dimension of separation in multidimensional shotgun proteomics analysis, with a potential for fully automated workflow.
最近，我们推出了一种在线多通道毛细管等电聚焦（OMJ-CIEF）分级仪，用于在电喷雾友好型溶液中对蛋白质和肽进行分级。在这项后续研究中，对分级仪的原始配置进行了改进，以提高分离的分辨率和重现性。主要改进包括使用分压器稳定通过该装置的电流，以及结合对剩余肽的重复聚焦进行肽区的逐步洗脱。此外，还开发了一种新算法，以便更准确地从实验数据中计算鉴定出的肽的pI值。未修饰肽的计算pI值与理论预测pI值的标准偏差平均为0.21 pH单位，这比基于标准分辨率凝胶的方法更准确。为了表征改进后装置的分析性能，将其应用于酵母蛋白质组消化物的pI分级，分为18个级分，收集的级分通过反相液相色谱-串联质谱进行分析。在20047个鉴定出的肽中，约37%仅在一个级分中检测到，27%在两个级分中检测到。平均而言，每个肽在2.4个级分中被发现。这些结果有力地表明，改进后的装置适用于多维鸟枪法蛋白质组学分析中的第一维分离，具有实现全自动工作流程的潜力。
