Blake Thomas A, Williams Tracie L, Pirkle James L, Barr John R
Biological Mass Spectrometry Laboratory, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-50, Atlanta, Georgia 30341, USA.
Anal Chem. 2009 Apr 15;81(8):3109-18. doi: 10.1021/ac900095h.
Using liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of deglycosylated and intact glycopeptides from tryptic digests of whole influenza virus, we determined that the six predicted N-linked glycosylation sites within the N-terminal ectodomain of hemagglutinin (HA) from three selected H5N1 strains are occupied. The use of selective sample preparation strategies, including solid-phase extraction (SPE) of glycopeptides via hydrazide capture chemistry as well as hydrophilic interaction liquid chromatography (HILIC), sufficiently reduced sample complexity to allow determination of occupied glycosylation sites. The specific amino acid sequence of the tryptic glycopeptides for the identified sites varied slightly among strains, but the overall locations of the occupied glycosylation sites were conserved in the protein sequence. We used this knowledge of glycosylation site occupation to examine the glycans attached to these occupied sites on HA for a reassortant H5N1 strain grown in embryonated chicken eggs. By applying mass spectrometry-based methodologies for examining glycosylation to the study of influenza virus proteins, we can better understand the effect that this post-translational modification has upon the virulence and antigenicity of emerging strains.
通过对全流感病毒胰蛋白酶消化产物中的去糖基化和完整糖肽进行液相色谱/串联质谱（LC/MS/MS）分析，我们确定了来自三种选定H5N1毒株的血凝素（HA）N端胞外域内六个预测的N-连接糖基化位点均被占据。使用选择性样品制备策略，包括通过酰肼捕获化学以及亲水相互作用液相色谱（HILIC）对糖肽进行固相萃取（SPE），充分降低了样品复杂性，从而能够确定被占据的糖基化位点。所鉴定位点的胰蛋白酶糖肽的特定氨基酸序列在毒株之间略有不同，但被占据的糖基化位点的总体位置在蛋白质序列中是保守的。我们利用这种关于糖基化位点占据情况的知识，来研究在鸡胚中生长的重组H5N1毒株HA上这些被占据位点所连接的聚糖。通过将基于质谱的糖基化检测方法应用于流感病毒蛋白研究，我们能够更好地理解这种翻译后修饰对新出现毒株的毒力和抗原性的影响。
