Use of multidimensional lectin affinity chromatography in differential glycoproteomics
Use of multidimensional lectin affinity chromatography in differential glycoproteomics
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DOI：
10.1021/ac048751x
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发表时间：
2005-05-01
影响因子：
7.4
通讯作者：
Regnier, FE
中科院分区：
文献类型：
--
作者：
Qiu, RQ;Regnier, FE
本文报道了对携带复杂双触角N -连接型、混合型和高甘露糖型寡糖的人血清糖蛋白之间唾液酸化相对程度的比较研究。通过将凝集素亲和选择与血清胰蛋白酶消化肽段的稳定同位素标记相结合进行比较。蛋白质水解后，将样品分开，并根据来源或后续的分离方法，用稳定同位素标记试剂进行差异乙酰化。用黑接骨木凝集素（SNA）制备的凝集素柱用于选择和比较含唾液酸糖肽的浓度。使用该方法定量的相对标准偏差为4%。利用这种方法，将一名正常人含唾液酸糖蛋白的浓度与大量正常人混合血清样本中的浓度进行了比较。结果发现，除了四五种糖蛋白外，所有糖蛋白的唾液酸化程度变化均小于2倍。进一步对糖蛋白内的唾液酸化程度进行了研究。用标记试剂的轻同位素形式标记的样品被应用于一组串联的凝集素柱，该组柱由伴刀豆球蛋白A（Con A）柱与SNA柱连接而成，用于选择连接在具有复杂双触角N -连接型、混合型和高甘露糖型聚糖的糖肽上的唾液酸。相反，用标记试剂的重同位素形式标记的样品仅应用于Con A凝集素柱，以选择含有复杂双触角N -连接型、混合型和高甘露糖型聚糖的糖肽，而不考虑唾液酸化。将如此选择的糖肽混合，用肽N -糖苷酶F去糖基化，并通过反相色谱（RPC）进行分离。然后用电喷雾质谱（ESI - MS）分析RPC分离组分。该方法的相对标准偏差为4%。除了一种糖肽外，所有鉴定出的糖肽都含有唾液酸。同位素异构体相对丰度相等的肽段被认为表明蛋白质母体在该特定糖基化位点是完全唾液酸化的。
This paper reports studies comparing the relative degree of sialylation among human serum glycoproteins carrying complex biantennary N-linked, hybrid, and high-mannose oligosaccharides. Comparisons were made by coupling lectin affinity selection with stable isotope coding of peptides from tryptic digests of serum. After proteolysis, samples were split and differentially acetylated with stable isotope coding agents according to either origin or the separation method by which they would be fractionated. A lectin column prepared from Sambucus nigra agglutinin (SNA) was used to select and compare the concentration of sialic acid containing glycopeptides. The relative standard deviation in quantification using this method was 4%. Using this method the concentration of sialic acid containing glycoproteins from a normal individual were compared to those in a pooled serum sample from a large number of normal individuals. It was found that sialylation varied less than 2-fold in all but four or five glycoproteins. Further studies were done on the degree of sialylation within glycoproteins. Samples labeled with the light isoform of the coding agent were applied to a set of serial lectin columns consisting of a concanavalin A (Con A) column coupled to an SNA column for selecting sialic acid appended to glycopeptides with complex biantennary N-linked, hybrid, and high-mannose glycans. In contrast, samples labeled with the heavy isoform of the coding agent were applied to a Con A lectin column alone to select glycopeptides containing complex biantennary N-linked, hybrid, and high-mannose glycans, without regard to sialylation. Glycopeptides thus selected were mixed, deglycosylated by PNGase F, and fractionated by reversed-phase chromatography (RPC). The RPC fractions were then analyzed by ESI-MS. The relative standard deviation of the method was 4%. All glycopeptides identified contained sialic acid except one. Peptides in which the relative abundance of isotopic isoforms was equal were considered to indicate that the protein parent was fully sialylated at that specific glycosylation site.