METHODS: We enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC-MS/MS.
RESULTS: Human albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated.
CONCLUSIONS: Albumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.
方法:我们富集了丰富的血清蛋白,以研究其N-连接糖基化,然后通过大小排阻或混合模式阴离子交换色谱进行胰蛋白酶消化和糖肽富集。通过富集糖肽的液相色谱-串联质谱(LC-MS/MS)评估了规范和非规范位点的糖基化。进行去糖基化分析以确认在非规范位点的N-连接糖基化。白蛋白衍生的糖肽被MS3片段化以确认连接的聚糖。对另外20个样品进行平行反应监测以验证这些发现。通过LC-MS/MS类似地分析牛和兔白蛋白衍生的糖肽。
结果:人白蛋白在两个非规范位点被N-糖基化,Asn68和Asn123。在带有四个复杂唾液酸化聚糖的两个位点检测到N-糖肽,并通过基于MS3的片段化和去糖基化研究进行验证。靶向质谱证实了20个另外的供体样品中的糖基化。最后,还发现牛和兔血清白蛋白中高度保守的Asn123被糖基化。
结论:白蛋白是一种具有保守的N-连接糖基化位点的糖蛋白,可能具有潜在的临床应用。