PDF(Pubmed)
Abstract：
BACKGROUND: Glycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated.
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.
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.
摘要：
背景:糖基化是一种酶催化的翻译后修饰,其不同于糖基化,并且存在于大多数血浆蛋白上。N-糖基化发生在天冬酰胺残基上，主要在规范的N-糖基化基序（Asn-X-Ser/Thr）内，尽管也报道了非规范的N-糖基化基序Asn-X-Cys/Val。白蛋白是血浆中最丰富的蛋白质，其糖基化在糖尿病中得到了充分研究。然而,白蛋白长期以来一直被认为是一种非糖基化的蛋白质，由于缺乏规范的基序。白蛋白包含两个非规范的N-糖基化基序，其中一个最近被报道为糖基化的。
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。
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。
