University Medical Center Hamburg-Eppendorf, Department of Clinical Chemistry, Mass Spectrometric Proteomics, Campus Forschung, N27 Room 00.008, Martinistr 52, 20246 Hamburg, Germany.
J Chromatogr A. 2010 May 7;1217(19):3321-9. doi: 10.1016/j.chroma.2009.10.028. Epub 2009 Oct 14.
It was the aim of this study to compare the performance of displacement chromatography with gradient elution chromatography both applied as the cation-exchange separation step for a proteome analysis in a bottom-up approach using multidimensional chromatography for the separation of tryptic peptides prior to their mass spectrometric analysis. The tryptic digest of the human Cohn fraction IV-4 served as a sample. For both chromatography modes commonly used operating parameters were chosen thus ensuring optimal separation results of equal sample amounts for each mode. All resulting fractions were analyzed with an HPLC-chip-LC-MS system. The eluate of the HPLC-chip column was ionized by electrospray ionization (ESI) and analyzed with an ion-trap mass spectrometer. For guaranteeing high confidence concerning the identity of the peptides, the mass spectrometric data were processed by different bioinformatic tools applying stringent criteria. By the displacement approach the total amount of identified proteins (78) was significantly higher than in the gradient mode (58). The results showed that displacement chromatography is a well suited alternative in comparison to gradient elution separation for analysis of proteomes via the bottom-up approach applying multidimensional chromatography, especially in those cases when larger quantities of proteins are available.
本研究旨在比较排阻色谱和梯度洗脱色谱的性能，这两种方法都应用于阳离子交换分离步骤，用于使用多维色谱在质谱分析之前对胰蛋白酶肽进行分离的自上而下的蛋白质组分析。人类 Cohn 级分 IV-4 的胰蛋白酶消化物用作样品。对于这两种色谱模式，选择了常用的操作参数，从而确保每种模式的等量样品都能获得最佳的分离效果。所有得到的馏分都用 HPLC 芯片 LC-MS 系统进行分析。HPLC 芯片柱的洗脱液通过电喷雾电离（ESI）进行电离，并通过离子阱质谱进行分析。为了保证对肽的身份有高度的置信度，质谱数据通过应用严格标准的不同生物信息学工具进行处理。通过置换方法，鉴定的蛋白质总量（78）明显高于梯度模式（58）。结果表明，与梯度洗脱分离相比，在多维色谱自上而下的蛋白质组分析中，置换色谱是一种很好的替代方法，特别是在有大量蛋白质的情况下。
