Faserl Klaus, Sarg Bettina, Sola Laura, Lindner Herbert H
Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria.
Institute of Chemistry of Molecular Recognition, National Research Council of Italy, Milano, Italy.
Proteomics. 2017 Nov;17(22). doi: 10.1002/pmic.201700310. Epub 2017 Oct 25.
In this study we demonstrate the potential of sequential injection of samples in capillary electrophoresis-mass spectrometry for rapid and sensitive proteome characterization of human lymphoblastic T-cells (line CCRF-CEM). Proteins were extracted, enzymatically digested, and the resulting peptides fractionated by RP-HPLC. Twenty fractions were thereafter analyzed by CE-MS within a single MS analysis. The CE-MS method was designed so that every 10 min a new fraction was injected into the CE system. Without any rinsing or equilibration steps we were able to generate a continuous stream of peptides feeding the mass analyzer. In 250 min, the total analysis time of a single sequential injection experiment, we were able to identify roughly 28 000 peptide sequences counting for 4800 proteins. These numbers could be increased to 62 000 peptides and more than 6100 proteins identified, when performing three experiments analyzing a total of 60 fractions, all within 12.5 h. We found that the electrophoretic mobility of peptides can be used to trace back peptides and assign them to the fraction they originate from.
在本研究中，我们展示了在毛细管电泳-质谱联用中顺序进样用于快速灵敏地表征人淋巴母细胞性T细胞（CCRF-CEM细胞系）蛋白质组的潜力。提取蛋白质，进行酶解，所得肽段通过反相高效液相色谱进行分离。之后在一次质谱分析中通过毛细管电泳-质谱联用对20个馏分进行分析。毛细管电泳-质谱联用方法的设计使得每隔10分钟就有一个新的馏分注入毛细管电泳系统。无需任何冲洗或平衡步骤，我们就能产生一股连续的肽流进入质谱分析仪。在一次顺序进样实验的250分钟总分析时间内，我们能够鉴定出约28000个肽序列，对应4800种蛋白质。当进行三次实验共分析60个馏分，且所有分析在12.5小时内完成时，这些数字可增加到鉴定出62000个肽和6100多种蛋白质。我们发现肽的电泳迁移率可用于追溯肽并将其归属于它们所源自的馏分。
