Department of Chemistry, Oklahoma State University, Stillwater, OK 74078-3071, United States.
Department of Chemistry, Oklahoma State University, Stillwater, OK 74078-3071, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Feb 1;1233:123992. doi: 10.1016/j.jchromb.2023.123992. Epub 2024 Jan 5.
In this study, a precursor carboxy-silica support was demonstrated in the immobilization of two different lectins, namely concanavalin A (Con A) and wheat germ agglutinin (WGA) for use in high performance lectin affinity chromatography (LAC) for the selective capturing and enrichment of glycoproteins from healthy/disease free and cancer human sera. The lectin columns thus obtained (i.e., Con A- and WGA-columns) showed no nonspecific interactions toward some chosen standard glycoproteins and non-glycoproteins. Both columns were shown in sub-glycoproteomics enrichment from human sera including disease free and adenocarcinoma cancer sera. The collected fractions were subjected to LC-MS/MS for identification of the captured glycoproteins, whereby the total number of identified proteins using Con A column from disease-free and cancer sera were 164 and 188, respectively while 133 and 103 proteins were identified in the fractions captured by the WGA column from disease-free and cancer sera samples, respectively. Differentially expressed proteins (DEPs) between the disease free and cancer sera in both the Con A and WGA column fractions were identified via the plot of the abundance vs. the protein ratio whereby the binary logarithm of average intensities of cancer and disease free sera were plotted against the binary logarithm of cancer/disease free sera ratios. The proteins that exhibit log 2 (cancer/healthy) ratio values greater than +2 and less than -2 in both categories are considered as DEPs. Furthermore, for visualization of the data arrangement, Q-Q scatterplot were also used whereby the binary logarithm of cancer serum was plotted against the binary logarithm of disease-free serum for both Con A and WGA. For Con A column, 28 up-regulated and 10 down regulated proteins were identified with a total of 38 DEPs while only two being non-glycoproteins. Furthermore, the up-regulated, and down regulated proteins recorded for WGA column are 14 and 6, respectively, totaling 20 proteins including 3 non-glycoproteins. Some of the non-specific binding to lectin are most likely due to protein-protein interactions.
在这项研究中，展示了一种前体羧基硅载体在固定两种不同的凝集素（即伴刀豆球蛋白 A（Con A）和麦胚凝集素（WGA））中的应用，用于从健康/无病和癌症人血清中选择性捕获和富集糖蛋白。由此获得的凝集素柱（即 Con A-和 WGA-柱）对一些选定的标准糖蛋白和非糖蛋白没有非特异性相互作用。这两个柱子都显示了从人血清中进行亚糖蛋白质组学富集的能力，包括无病和腺癌癌症血清。收集的馏分进行 LC-MS/MS 分析，以鉴定捕获的糖蛋白，使用 Con A 柱从无病和癌症血清中鉴定的总蛋白数分别为 164 和 188，而使用 WGA 柱从无病和癌症血清中鉴定的蛋白数分别为 133 和 103。通过对 Con A 和 WGA 柱馏分中无病和癌症血清之间的差异表达蛋白（DEPs）进行图谱分析，通过对丰度与蛋白比的作图来鉴定差异表达蛋白，其中癌症和无病血清的平均强度的对数值与癌症/无病血清比值的对数值作图。在这两个类别中，log 2（癌症/健康）比值大于+2 且小于-2 的蛋白被认为是 DEPs。此外，为了可视化数据排列，还使用了 Q-Q 散点图，其中 Con A 和 WGA 中均将癌症血清的对数值与无病血清的对数值作图。对于 Con A 柱，鉴定出 28 个上调和 10 个下调蛋白，共有 38 个 DEPs，其中只有 2 个是非糖蛋白。此外，WGA 柱记录的上调和下调蛋白分别为 14 和 6，共 20 个蛋白，其中包括 3 个非糖蛋白。一些与凝集素的非特异性结合很可能是由于蛋白质-蛋白质相互作用。
