Department of Chemical Engineering, University of the West Indies, Trinidad and Tobago.
Biointerphases. 2011 Sep;6(3):135. doi: 10.1116/1.3622347.
Although previous studies have demonstrated that TOF-SIMS is a powerful method for the characterization of adsorbed proteins due to its specificity and surface sensitivity, it was unclear from earlier work whether the differences between proteins observed on uniform flat surfaces were large enough to facilitate clear image contrast between similar proteins in small areas on topographically complex samples that are more typical of biological tissues. The goal of this study was to determine whether Bi(3) (+) could provide sufficiently high sensitivity to provide clear identification of the different proteins in an image. In this study, 10 μm polystyrene microspheres were adsorbed with one of three different proteins, human serum albumin (HSA), bovine serum albumin (BSA), and hemoglobin. Spheres coated with HSA were then mixed with spheres coated with either BSA (a very similar protein) or hemoglobin (a dramatically different protein), and deposited on silicon substrates. Fluorescent labeling was used to verify the SIMS results. With maximum autocorrelation factors (MAF) processing, images showed clear contrast between both the very different proteins (HSA and hemoglobin) and the very similar proteins (HSA and BSA). Similar results were obtained with and without the fluorescent labels. MAF images were calculated using both the full spectrum and only characteristic amino acid fragments. Although better image contrast was obtained using the full spectrum, differences between the spheres were still evident when only the amino acid fragments were included in the analysis, suggesting that we are truly observing differences between the proteins themselves. These results demonstrate that TOF-SIMS, with a Bi(3) (+) primary ion, is a powerful technique for characterizing interfacial proteins not only on large uniform surfaces, but also with high spatial resolution on the topographically complex samples typical in biological analysis.
虽然先前的研究已经表明，TOF-SIMS 是一种强大的方法，用于表征吸附蛋白，因为其特异性和表面灵敏度，但从早期的工作中不清楚在均匀平坦表面上观察到的蛋白质之间的差异是否足够大，以促进在地形复杂的样品上的小区域中相似蛋白质之间的清晰图像对比，这些样品更典型的是生物组织。本研究的目的是确定 Bi(3) (+) 是否能够提供足够高的灵敏度，以提供图像中不同蛋白质的清晰识别。在这项研究中，10 μm 聚苯乙烯微球吸附了三种不同蛋白质中的一种，即人血清白蛋白（HSA）、牛血清白蛋白（BSA）和血红蛋白。然后将涂覆有 HSA 的球体与涂覆有 BSA（非常相似的蛋白质）或血红蛋白（差异非常大的蛋白质）的球体混合，并沉积在硅衬底上。荧光标记用于验证 SIMS 结果。通过最大自相关因子（MAF）处理，图像显示出非常不同的蛋白质（HSA 和血红蛋白）和非常相似的蛋白质（HSA 和 BSA）之间的清晰对比。有和没有荧光标记都得到了类似的结果。使用全光谱和仅特征氨基酸片段计算 MAF 图像。虽然使用全光谱可以获得更好的图像对比度，但当仅包括氨基酸片段进行分析时，球体之间的差异仍然明显，这表明我们真正观察到的是蛋白质本身之间的差异。这些结果表明，TOF-SIMS 与 Bi(3) (+) 初级离子一起，不仅是一种强大的技术，用于表征大的均匀表面上的界面蛋白质，而且还具有生物分析中典型的地形复杂样品的高空间分辨率。
