QUANTITATIVE MASS SPECTROMETRY TO PROBE FIBRINOGEN CONFORMATIONS ON BIOMATERIALS

描述(申请人提供)：我们将应用化学标记和质谱学方法来研究蛋白质在生物材料上的吸附。我们相信，使用这些方法，我们可以将蛋白质吸附后发生的构象变化指定到蛋白质上的特定位置。我们最近发表的一项研究表明，随着溶液浓度的降低，纤维蛋白原上赖氨酸基团的化学标记增加。现在，我们希望使用定量质谱学和化学标记技术来进一步探索纤维蛋白原在两种生物材料--PET和PTFE--吸附后的构象变化。定量质谱学很难用蛋白质来实现。我们在华盛顿大学的合作者已经展示了一种非常有希望的技术，它可以减少与串联质谱仪进行蛋白质定量相关的误差。吸附蛋白质的化学标记检测蛋白质构象变化和定量质谱学相结合的方法，可能会推动蛋白质在生物材料上的吸附研究的进展。新技术的使用将被用于回答有关纤维蛋白原吸附后构象变化对血小板与生物材料粘附性的影响的问题。我们将证明：目标1：纤维蛋白原在生物材料上的吸附导致赖氨酸和酪氨酸基团在促进生物活性的部位附近的溶剂暴露发生变化。目的2：这些部位的溶剂暴露程度与血小板与吸附的纤维蛋白原的粘附性有关。公共卫生相关性：针对人工植入材料的生物反应限制了医疗设备的使用，如血管移植物、血管内支架和许多其他与流动的血液接触的设备。这种反应的主要媒介是蛋白纤维蛋白原，它能吸附到材料上，并支持血小板和白细胞的黏附。拟议的项目将探索导致生物反应的吸附纤维蛋白原的构象变化，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.