QUANTITATIVE MASS SPECTROMETRY TO PROBE FIBRINOGEN CONFORMATIONS ON BIOMATERIALS

描述（由申请人提供）：我们将应用化学标记和质谱方法来研究蛋白质在生物材料上的吸附。我们相信，通过这些方法，我们可以将吸附后蛋白质内部发生的构象变化分配到蛋白质上的特定位点。我们最近发表的一项研究表明，当溶液浓度降低时，纤维蛋白原上赖氨酸基团的化学标记增加。我们现在希望使用定量质谱结合化学标记技术来进一步探索纤维蛋白原在吸附到PET和PTFE两种生物材料时构象的变化。定量质谱分析在蛋白质中很难实现。我们在华盛顿大学的合作者已经展示了一种技术，它在减少串联质谱测定蛋白质定量的错误方面非常有前途。将吸附蛋白质的化学标记检测蛋白质构象的变化与定量质谱法相结合，可能会导致蛋白质在生物材料上吸附的研究取得进展。新技术的使用将直接用于回答有关纤维蛋白原吸附后构象变化对血小板粘附生物材料的影响的问题。我们将证明：目标1：纤维蛋白原对生物材料的吸附会导致溶剂暴露在促进生物活性位点附近的赖氨酸和酪氨酸基团的变化。目的2：这些部位的溶剂暴露程度与血小板粘附在纤维蛋白原上有关。公共卫生相关性：对人工植入材料产生的生物反应限制了医疗器械的使用，如血管移植物、血管内支架和许多其他与流动血液接触的器械。反应的主要介质是蛋白质纤维蛋白原，它吸附物质并支持血小板和白细胞的粘附。该项目将探索吸附纤维蛋白原的构象变化导致的生物反应，这将使我们更好地了解材料的生物相容性。

DESCRIPTION (provided by applicant): We will apply chemical labeling and mass spectrometric methods to the study of protein adsorption on biomaterials. We believe that with these methods, we can assign the conformational changes that occur within proteins following adsorption to specific sites on the protein. We have recently published a study that demonstrated that the chemical labeling of lysine groups on fibrinogen increased when the solution concentration decreased. We now wish to use quantitative mass spectrometry combined with the chemical labeling technique to further explore changes in fibrinogen conformations upon adsorption to two biomaterials, PET and PTFE. Quantitative mass spectrometry is difficult to achieve with proteins. Our collaborators at Washington University have demonstrated a technique that is quite promising in reducing errors associated with protein quantification by tandem mass spectrometry. The combination of methods, chemical labeling of adsorbed proteins to detect changes in protein conformation and quantitative mass spectrometry, may lead to advances in the study of protein adsorption on biomaterials. The use of the new techniques will be directed towards answering questions about the impact of fibrinogen's post-adsorptive conformational changes on the adhesion of platelets to biomaterials. We will demonstrate that: Aim 1: Adsorption of fibrinogen to biomaterials leads to changes in the solvent exposure of lysine and tyrosine groups near sites that promote biological activity. Aim 2: The degree of solvent exposure at these sites correlates with platelet adhesion to adsorbed fibrinogen. PUBLIC HEALTH RELEVANCE: The biological response that is mounted against artificial implanted materials constrains the use of medical devices such as vascular grafts, endovascular stents and many other devices that are in contact with flowing blood. A major mediator of the response is the protein fibrinogen, which adsorbs to materials and supports platelet and leukocyte adhesion. The proposed project will probe the conformational changes in adsorbed fibrinogen that lead to biological responses, which will allow us to better understand the biocompatibility of materials.