Prior to the identification of proteins for proteomics analysis in human cells, separation of fluorogenic derivatized proteins with a fluorogenic reagent, 7‐chloro‐N‐[2‐(dimethylamino)ethyl]‐2,1,3‐benzoxadiazole‐4‐sulfonamide, has typically been performed by using a conventional reversed‐phase HPLC column. However, the number of proteins in human cells (HepaRG) that are separated by this conventional approach is limited to approximately 500. In this study, a nanoflow liquid chromatography system with an evaluated phenyl‐bonded monolithic silica capillary column (0.1 mm i.d., 700 mm length) was used to increase the number of separated fluorogenic derivatized proteins. This system was used to separate derivatized human cell proteins (K562) and yeast (Saccharomyces cerevisiae) proteins as model cell proteomes. More than 1,300 protein peaks were separated/detected from both cell proteomes. We present a straightforward comparison of multiple separation profiles using a novel chromatogram display approach, termed the “spiderweb” chromatogram. In addition, to validate that the detected peaks are derived from proteins, a mass spectrometer was connected to the capillary column and deconvolution of the obtained mass spectra was performed. Furthermore, different molecular weight distribution profiles of the expressed proteins were observed between the two cell proteomes.

中文翻译：

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苯基键合的整体硅胶毛细管柱液相色谱分离和荧光衍生的完整蛋白的检测

在鉴定用于人体细胞蛋白质组学分析的蛋白质之前，请先使用荧光试剂7-氯-N- [2-（二甲基氨基）乙基] -2,1,3-苯并恶二唑-4-磺酰胺分离出荧光衍生的蛋白质，通常使用常规的反相HPLC色谱柱进行分析。但是，通过这种常规方法分离的人细胞（HepaRG）中的蛋白质数量限制为大约500种。在这项研究中，纳米流液相色谱系统带有经过评估的苯基键合整体硅胶毛细管柱（内径0.1 mm，700长度（mm））用于增加分离的荧光衍生蛋白的数量。该系统用于分离衍生的人细胞蛋白（K562）和酵母（酿酒酵母）作为模型细胞蛋白质组。从这两种细胞蛋白质组中分离/检测到1,300多个蛋白质峰。我们使用一种称为“ spiderweb”色谱的新型色谱显示方法，对多种分离曲线进行了直接比较。另外，为了验证检测到的峰是从蛋白质中衍生出来的，将质谱仪连接到毛细管柱上，并对获得的质谱进行解卷积。此外，在两个细胞蛋白质组之间观察到表达的蛋白质的不同分子量分布图。