Integration of On-Column Chemical Reactions in Protein Characterization by Liquid Chromatography/Mass Spectrometry: Cross-Path Reactive Chromatography
Integration of On-Column Chemical Reactions in Protein Characterization by Liquid Chromatography/Mass Spectrometry: Cross-Path Reactive Chromatography
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DOI：
10.1021/acs.analchem.7b04328
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发表时间：
2018-01-16
影响因子：
7.4
通讯作者：
Kaltashov, Igor A.
中科院分区：
文献类型：
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作者：
Pawlowski, Jake W.;Carrick, Ian;Kaltashov, Igor A.
通过质谱法（MS）分析复杂蛋白质通常需要对其共价结构进行某些化学修饰（例如，二硫键的还原），在MS或MS/MS分析之前进行。传统上，这些化学反应在离线模式下发生，以允许在MS测量之前从蛋白质溶液中去除过量的试剂（其中大部分试剂干扰MS测量并降低分析信号）。除了分析时间的显著增加之外，如果修饰不利地影响蛋白质的稳定性，例如使其易于聚集，则化学反应可能导致蛋白质的部分或全部损失。在这项工作中，我们提出了一种新的方法来解决这个问题，通过进行化学反应在线使用的反应色谱方案上的尺寸排阻色谱（SEC）平台与MS检测。这通过使用交叉路径反应方案来实现，即，通过延迟蛋白质注入SEC柱（相对于含有二硫化物还原剂的试剂塞的注入），这允许化学反应在柱内进行有限的（和精确控制的）时间段，同时两个塞在柱内重叠。还原的蛋白质与未消耗的试剂分开洗脱，从而避免ESI中的信号抑制，并实现灵敏的MS检测。该新方法用于在线将二硫键连接的四聚体物质还原为单体单元后，在全蛋白水平上测量血浆蛋白结合珠蛋白的岩藻糖基化水平。自上而下的片段化的含二硫键的蛋白质的可行性也证明了使用firmicroglobulin和单克隆抗体（mAb）。新的在线技术既稳健又通用，因为交叉路径方案可以很容易地扩展到在单个实验中包括多个反应（如本工作中通过在色谱柱上氧化标记mAb，然后还原其二硫键和MS分析分子每条链内的氧化程度所证明的）。
Profiling of complex proteins by means of mass spectrometry (MS) frequently requires that certain chemical modifications of their covalent structure (e.g., reduction of disulfide bonds), be carried out prior to the MS or MS/MS analysis. Traditionally, these chemical reactions take place in the off-line mode to allow the excess reagents (the majority of which interfere with the MS measurements and degrade the analytical signal) to be removed from the protein solution prior to MS measurements. In addition to a significant increase in the analysis time, chemical reactions may result in a partial or full loss of the protein if the modifications adversely affect its stability, e.g, making it prone to aggregation. In this work we present a new approach to solving this problem by carrying out the chemical reactions online using the reactive chromatography scheme on a size exclusion chromatography (SEC) platform with MS detection. This is achieved by using a cross-path reaction scheme, i.e., by delaying the protein injection onto the SEC column (with respect to the injection of the reagent plug containing a disulfide reducing agent), which allows the chemical reactions to be carried out inside the column for a limited (and precisely controlled) period of time, while the two plugs overlap inside the column. The reduced protein elutes separately from the unconsumed reagents, allowing the signal suppression in ESI to be avoided and enabling sensitive MS detection. The new method is used to measure fucosylation levels of a plasma protein haptoglobin at the whole protein level following online reduction of disulfide linked tetrameric species to monomeric units. The feasibility of top-down fragmentation of disulfide-containing proteins is also demonstrated using firmicroglobulin and a monoclonal antibody (mAb). The new online technique is both robust and versatile, as the cross-path scheme can be readily expanded to include multiple reactions in a single experiment (as demonstrated in this work by oxidatively labeling mAb on the column, followed by reduction of its disulfide bonds and MS analysis of the extent of oxidation within each chain of the molecule).