Ito Hiroki, Takegawa Yasuhiro, Deguchi Kisaburo, Nagai Shinji, Nakagawa Hiroaki, Shinohara Yasuro, Nishimura Shin-Ichiro
Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
Rapid Commun Mass Spectrom. 2006;20(23):3557-65. doi: 10.1002/rcm.2761.
Mass spectrometric analyses of various N-glycans binding to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on collision-induced dissociation (CID) MS(n) spectra acquired by electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) in the positive- and negative-ion modes has been proposed as a direct method of assigning N-glycans without releasing them from N-glycopeptides. In the positive-ion mode of this approach, the MS(2) spectrum of N-glycopeptide was acquired so that a glycoside-bond cleavage occurs in the chitobiose residue (i.e., GlcNAcbeta1-4GlcNAc, GlcNAc: N-acetylglucosamine) attached to asparagine (N), and two charges on the M+H+Na precursor ion are shared with both of the resulting fragments. These fragments are sodiated B(n)-type fragment ions of oligosaccharide (N-glycan) and a protonated peptide ion retaining one GlcNAc residue on the asparagine (N) residue. The structure of N-glycan was assigned by comparing MS(3) spectra derived from both the sodiated B(n)-type fragment ions of N-glycopeptide and the PA (2-aminopyridine) N-glycan standard (i.e., MS(n) spectral matching). In a similar manner, the structural assignment of sialylated N-glycan was performed by employing the negative-ion CID MS(n) spectra of deprotonated B(n)-type fragment ions of N-glycopeptide and the PA N-glycan standard. The efficacy of this approach was tested with chicken egg yolk glycopeptides with a neutral and a sialylated N-glycan, and human serum IgG glycopeptides with neutral N-glycan isomers. These results suggest that the approach based on MS(n) spectral matching is useful for the direct and simple structural assignment of neutral and sialylated N-glycans of glycopeptides.
对与蛋白质和肽结合的各种N-聚糖进行质谱分析，对于阐明其生物学作用非常必要。一种基于电喷雾电离线性离子阱飞行时间质谱（ESI-LIT-TOFMS）在正离子和负离子模式下获得的碰撞诱导解离（CID）MS(n)谱的方法，已被提出作为一种直接分配N-聚糖的方法，而无需将它们从N-糖肽中释放出来。在该方法的正离子模式下，获取N-糖肽的MS(2)谱，使得连接到天冬酰胺（N）上的壳二糖残基（即GlcNAcbeta1-4GlcNAc，GlcNAc：N-乙酰葡糖胺）中发生糖苷键裂解，并且M+H+Na前体离子上的两个电荷与产生的两个片段共享。这些片段是寡糖（N-聚糖）的钠化B(n)型片段离子和在天冬酰胺（N）残基上保留一个GlcNAc残基的质子化肽离子。通过比较来自N-糖肽的钠化B(n)型片段离子和PA（2-氨基吡啶）N-聚糖标准品的MS(3)谱（即MS(n)谱匹配）来确定N-聚糖的结构。以类似的方式，通过采用N-糖肽的去质子化B(n)型片段离子和PA N-聚糖标准品的负离子CID MS(n)谱来进行唾液酸化N-聚糖的结构鉴定。用具有中性和唾液酸化N-聚糖鸡卵黄糖肽以及具有中性N-聚糖异构体的人血清IgG糖肽测试了该方法的有效性。这些结果表明，基于MS(n)谱匹配的方法对于糖肽中中性和唾液酸化N-聚糖的直接和简单结构鉴定是有用的。
