High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery
High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery
复制标题
DOI：
10.1002/elps.201100341
复制
发表时间：
2011-12-01
期刊：
影响因子：
2.9
通讯作者：
Hill, Michelle M.
中科院分区：
文献类型：
--
作者：
Choi, Eunju;Loo, Dorothy;Hill, Michelle M.
蛋白糖基化的改变发生在许多疾病的发生和发展过程中，因此糖组学和糖蛋白组学已成为发现糖生物标志物的重要工具。高通量聚糖分析现在可以实现与MS-based技术的最新发展。为了鉴定和快速监测血清蛋白的糖基化变化，我们开发了一种半自动的高通量糖蛋白生物标志物发现平台，称为凝集素磁珠阵列耦合串联质谱（LeMBA-MS），其中包括(i)使用微孔板格式的20个单个凝集素包被磁珠进行有效的单步血清糖蛋白分离，（ii）在磁珠上胰蛋白酶消化，以及（iii）具有凝集素排除列表的纳米lc -MS/MS。通过使用合适的序列数据库，LeMBA-MS可以检测不同物种的糖基化变化。通过将已知量的卵清蛋白滴定到血清样品中，我们报告了使用豆豆蛋白a偶联珠粒的LeMBA-MS的纳摩尔灵敏度和线性响应。神经氨酸酶处理导致与唾液酸结合凝集素的结合减少。有趣的是，我们发现去脱醛化导致接触珠蛋白和血凝素与甘露糖特异性凝集素的结合增加，这表明了识别凝集素结合特征的重要性。高通量LeMBA-MS生成糖基化特征将促进糖生物标志物的发现。LeMBA可以耦合到下游检测平台进行验证，使其成为一个真正通用的平台。
Alterations in protein glycosylation occur during development and progression of many diseases, hence glycomics and glycoproteomics have emerged as important tools in glycobiomarker discovery. High-throughput glycan profiling can now be achieved with the recent developments in MS-based techniques. To enable identification and rapid monitoring of glycosylation changes in serum proteins, we developed a semi-automated high-throughput glycoprotein biomarker discovery platform termed lectin magnetic bead array-coupled tandem mass spectrometry (LeMBA-MS) which includes (i) effective single-step serum glycoprotein isolation using a panel of 20 individual lectin-coated magnetic beads in microplate format, (ii) on-bead trypsin digestion, and (iii) nanoLC-MS/MS with lectin exclusion list. With use of appropriate sequence databases, LeMBA-MS can detect glycosylation changes regardless of the species. By spiking known amounts of titrated ovalbumin to a serum sample, we report nanomolar sensitivity, and linearity of response of LeMBA-MS using concanavalin A-coupled beads. Neuraminidase treatment led to reduction of binding to sialic acid-binding lectins. Interestingly, we found that desialylation caused increased binding of haptoglobin and hemopexin to mannose-specific lectins, pointing to the importance of identifying a signature of lectin-binding. High-throughput LeMBA-MS to generate glycosylation signatures will facilitate glycobiomarker discovery. LeMBA can be coupled to down-stream detection platforms for validation, making it a truly versatile platform.