Qiu Ruiqing, Regnier Fred E
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Anal Chem. 2005 Nov 15;77(22):7225-31. doi: 10.1021/ac050554q.
This study describes a simple and efficient approach for comparative analysis of sialylated glycoforms of proteins containing differentially branched complex-type glycans. The analytical protocol is based on glycopeptide selection from tryptic digests with serial lectin affinity chromatography (SLAC), quantification with global internal standard technology, fractionation of deglycosylated peptides with reversed-phase chromatography, and peptide sequencing with tandem mass spectrometry. Fractionation of complex tri- and tetraantennary N-linked glycoforms from biantennary N-linked glycoforms bearing terminal sialic acid residues was achieved using a set of serial lectin columns with immobilized Sambucus nigra agglutinin and concanavalin A. These two fractions from the affinity selection were differentially labeled, mixed, and then deglycosylated with the enzyme PNGase F. The deglycosylated sample was further fractionated by reversed-phase chromatography and analyzed by electrospray ionization mass spectrometry. The SLAC strategy was applied to tryptic digests of human serum, and it was found that most sialylated glycopeptides identified carry more biantennary glycans than tri- and tetraantennary glycans, and the relative amount of biantennary glycan versus tri- and tetraantennary glycans was different at separate glycosylation sites within the same glycoprotein.
本研究描述了一种简单有效的方法，用于对含有不同分支的复合型聚糖的蛋白质的唾液酸化糖型进行比较分析。该分析方案基于通过串联凝集素亲和色谱法（SLAC）从胰蛋白酶消化物中选择糖肽，采用全局内标技术进行定量，用反相色谱法对去糖基化肽进行分级分离，以及用串联质谱法进行肽测序。使用一组固定有黑接骨木凝集素和伴刀豆球蛋白A的串联凝集素柱，实现了从带有末端唾液酸残基的双天线N-连接糖型中分离复杂的三天线和四天线N-连接糖型。对亲和选择得到的这两个级分进行差异标记、混合，然后用PNGase F酶进行去糖基化。去糖基化后的样品通过反相色谱法进一步分级分离，并用电喷雾电离质谱法进行分析。将SLAC策略应用于人血清的胰蛋白酶消化物，发现鉴定出的大多数唾液酸化糖肽携带的双天线聚糖比三天线和四天线聚糖更多，并且在同一糖蛋白内的不同糖基化位点，双天线聚糖与三天线和四天线聚糖的相对量也不同。
