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Displacement chromatography for proteomic applications.

机译：用于蛋白质组学的置换色谱。

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摘要

Ion exchange displacement chromatography, chromatographic modeling, and mass spectrometry were employed to develop a new proteomics analysis platform which has the potential to achieve increased sensitivity and dynamic range for complex proteomic applications. Studies were carried out using high affinity, low molecular mass displacers for the ion exchange displacement of proteins and peptides to determine the limits of this approach. Model protein mixtures were employed to study the effects of varying displacer concentration and feed dynamic range. Results indicated that higher displacer concentrations produced elevated product concentrations at the price of reduced yields. Further, high affinity displacers employed at relatively low concentrations resulted in high resolution separations amenable to the identification of low abundance proteins. Good agreement was obtained between simulations carried out using a Steric Mass Action (SMA) based chromatographic model and the experiments performed. Parametric simulation studies were then carried out using the SMA model to investigate the concentration effects and enrichment factors observed over a wide range of conditions. Proof-of-concept column separations were carried out which illustrated that ion exchange displacement chromatography could be employed for protein pre-fractionation of complex model mixtures. This work demonstrated that displacement chromatography could be employed for trace component detection of proteins not found in feed mixtures, and that these displacement separations were amenable to subsequent mass spectrometry analysis without further processing since these displacement separations could be performed with minimal or no salt in the carrier. Displacement separations were also performed on the soluble proteins obtained from an E. coli cell lysate. These analyses indicated that proteins not detected in the feed mixture (due to their low concentrations) were able to be identified in the collected displacement effluent fractions. Off-line peptide fractionations using traditional and carrier displacement chromatography were also performed. These separations yielded encouraging results, both in terms of the grouping of peptides into various fractions having similar resin affinity and in terms of the concentration and enrichment of the peptides observed. Finally, a salt-free ion exchange process was developed to permit direct coupling of ion exchange displacement separations with on-line mass spectrometry analysis. This an important result since ion exchange chromatography and mass spectrometry typically cannot be directly coupled due to the high salt concentrations required for solute elution (for elution separations) and the inability of mass spectrometers to handle this salt concentration. The results presented in this thesis demonstrate the ability of ion exchange displacement chromatography to focus trace proteins into concentrated and fractionated zones. This body of work may have significant implications for the implementation of displacement chromatography for separations where trace enrichment is critical such as proteomic applications.
机译：离子交换置换色谱，色谱建模和质谱技术被用于开发新的蛋白质组学分析平台，该平台有可能为复杂的蛋白质组学应用提供更高的灵敏度和动态范围。使用高亲和力，低分子量置换剂进行了蛋白质和肽的离子交换置换研究，以确定该方法的局限性。模型蛋白质混合物用于研究不同的浮选剂浓度和饲料动态范围的影响。结果表明，较高的置换剂浓度以降低的收率为代价产生了较高的产物浓度。此外，以相对低的浓度使用的高亲和力置换剂导致适于分离低丰度蛋白质的高分辨率分离。使用基于Steric Mass Action（SMA）的色谱模型进行的模拟与所进行的实验之间获得了良好的一致性。然后使用SMA模型进行参数模拟研究，以研究在各种条件下观察到的浓度效应和富集因子。进行了概念验证的柱分离，这表明离子交换置换色谱可用于复杂模型混合物的蛋白质预分离。这项工作证明了置换色谱法可用于检测饲料混合物中未发现的蛋白质的痕量成分，并且这些置换分离法无需进一步处理即可用于后续的质谱分析，因为这些置换分离法可在样品中加入极少盐或无盐的条件下进行。载体。还对获自大肠杆菌细胞裂解物的可溶性蛋白质进行了置换分离。这些分析表明，在饲料混合物中未检测到的蛋白质（由于其浓度低）能够在收集的置换废液馏分中鉴定出来。还使用传统色谱法和载流子置换色谱法进行了离线肽分离。这些分离产生令人鼓舞的结果，无论是将肽分组为具有相似树脂亲和力的各种馏分，还是观察到的肽的浓度和富集。最后，开发了一种无盐离子交换工艺，可将离子交换置换分离与在线质谱分析直接耦合。这是一个重要的结果，因为由于溶质洗脱（用于洗脱分离）所需的高盐浓度以及质谱仪无法处理此盐浓度，因此离子交换色谱和质谱通常无法直接耦合。本文提出的结果证明了离子交换置换色谱法能够将痕量蛋白质聚焦到浓缩和分级区域中。这项工作可能对置换色谱法的实施具有重大意义，因为置换分离法对痕量富集至关重要，例如蛋白质组学应用。

著录项

  • 作者

    Evans, Steven T.;

  • 作者单位

    Rensselaer Polytechnic Institute.;

  • 授予单位 Rensselaer Polytechnic Institute.;
  • 学科 Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 293 p.
  • 总页数 293
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:24

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