On-plate glycoproteins/glycopeptides selective enrichment and purification based on surface pattern for direct MALDI MS analysis.
On-plate glycoproteins/glycopeptides selective enrichment and purification based on surface pattern for direct MALDI MS analysis.
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DOI：
10.1039/c3an00107e
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发表时间：
2013-04
期刊：
影响因子：
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通讯作者：
Zhoufang Zeng;Yandong Wang;Xinhua Guo;Ling Wang;Nan Lu
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文献类型：
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作者：
Zhoufang Zeng;Yandong Wang;Xinhua Guo;Ling Wang;Nan Lu
本文提出了一种在表面图案化样品支持物上实现糖蛋白/糖肽选择性富集和纯化的新方法，该样品支持物由疏水性外层（F-SAM）和内部硼酸修饰的金微点（900 μm）组成。在沉积时，样品溶液首先通过疏水外层的排斥而浓缩在小区域中，然后糖蛋白/糖肽通过硼酸共价结合在内层中而被选择性地捕获。然而，非糖基化蛋白质/肽或高浓度盐在用碱性溶液冲洗后被去除。结果，检测灵敏度比使用不锈钢MALDI板时提高了一个数量级。使用表面图案化样品支持物，即使在过量存在的非糖基化蛋白质/肽（比糖蛋白/糖肽多10倍）的干扰下，也可以检测糖蛋白/糖肽。同时，即使在尿素（1 M）、NaCl（1 M）或NH 4 HCO 3（200 mM）存在下也可获得高质量的质谱。因此，该技术可应用于复杂蛋白质组研究中低丰度糖蛋白/糖肽的高通量分析。
In this paper, a novel method has been proposed to achieve selective enrichment and purification of glycoproteins/glycopeptides on a surface patterned sample support, which consists of a hydrophobic outer layer (F-SAM) and an internal boronic acid-modified gold microspot (900 μm). Upon deposition, the sample solution is firstly concentrated in a small area by repulsion of the hydrophobic outer layer, and then the glycoproteins/glycopeptides are selectively captured through boronic acid covalently binding in the inner layer. However, the non-glycosylated proteins/peptides or high concentration salts are removed after rinsing with alkaline solution. As a result, the detection sensitivity is improved by an order of magnitude greater than when using a stainless steel MALDI plate. With surface patterned sample support, the glycoproteins/glycopeptides can be detected even under interference from the excessive existing non-glycosylated proteins/peptides (10 times more than glycoproteins/glycopeptides). Simultaneously, high-quality mass spectra can be obtained even in the presence of urea (1 M), NaCl (1 M), or NH4HCO3 (200 mM). Therefore, this novel technique may be applied to high-throughput analysis of low-abundance glycoproteins/glycopeptides in complicated proteome research.