Lee Richard, Ptolemy Adam S, Niewczas Liliana, Britz-McKibbin Philip
Department of Chemistry, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
Anal Chem. 2007 Jan 15;79(2):403-15. doi: 10.1021/ac061780i.
Characterization of unknown low-abundance metabolites in biological samples is one the most significant challenges in metabolomic research. In this report, an integrative strategy based on capillary electrophoresis-electrospray ionization-ion trap mass spectrometry (CE-ESI-ITMS) with computer simulations is examined as a multiplexed approach for studying the selective nutrient uptake behavior of E. coli within a complex broth medium. On-line sample preconcentration with desalting by CE-ESI-ITMS was performed directly without off-line sample pretreatment in order to improve detector sensitivity over 50-fold for cationic metabolites with nanomolar detection limits. The migration behavior of charged metabolites were also modeled in CE as a qualitative tool to support MS characterization based on two fundamental analyte physicochemical properties, namely, absolute mobility (muo) and acid dissociation constant (pKa). Computer simulations using Simul 5.0 were used to better understand the dynamics of analyte electromigration, as well as aiding de novo identification of unknown nutrients. There was excellent agreement between computer-simulated and experimental electropherograms for several classes of cationic metabolites as reflected by their relative migration times with an average error of <2.0%. Our studies revealed differential uptake of specific amino acids and nucleoside nutrients associated with distinct stages of bacterial growth. Herein, we demonstrate that CE can serve as an effective preconcentrator, desalter, and separator prior to ESI-MS, while providing additional qualitative information for unambiguous identification among isobaric and isomeric metabolites. The proposed strategy is particularly relevant for characterizing unknown yet biologically relevant metabolites that are not readily synthesized or commercially available.
生物样品中未知低丰度代谢物的表征是代谢组学研究中最重大的挑战之一。在本报告中，一种基于毛细管电泳-电喷雾电离-离子阱质谱联用（CE-ESI-ITMS）并结合计算机模拟的综合策略被作为一种多重方法进行研究，用于考察大肠杆菌在复杂肉汤培养基中的选择性营养摄取行为。通过CE-ESI-ITMS进行在线样品预浓缩并脱盐，无需离线样品预处理，以便将阳离子代谢物的检测灵敏度提高50倍以上，达到纳摩尔检测限。带电代谢物的迁移行为也在CE中进行建模，作为一种定性工具，以基于两种基本分析物物理化学性质，即绝对迁移率（μo）和酸解离常数（pKa）来支持质谱表征。使用Simul 5.0进行计算机模拟，以更好地理解分析物电迁移的动力学，以及辅助对未知营养物进行从头鉴定。几类阳离子代谢物的计算机模拟电泳图与实验电泳图之间存在极好的一致性，这通过它们的相对迁移时间得以体现，平均误差<2.0%。我们的研究揭示了与细菌生长不同阶段相关的特定氨基酸和核苷营养物的差异摄取。在此，我们证明CE可在ESI-MS之前作为一种有效的预浓缩器、脱盐器和分离器，同时为等压和同分异构代谢物之间的明确鉴定提供额外的定性信息。所提出的策略对于表征尚未容易合成或无法从商业途径获得的未知但具有生物学相关性的代谢物尤为重要。
