Determination and characterization of site-specific N-glycosylation using MALDI-Qq-TOF tandem mass spectrometry: Case study with a plant protease
Determination and characterization of site-specific N-glycosylation using MALDI-Qq-TOF tandem mass spectrometry: Case study with a plant protease
复制标题
DOI：
10.1021/ac0512711
复制
发表时间：
2006-02-15
影响因子：
7.4
通讯作者：
Ens, W
中科院分区：
文献类型：
--
作者：
Bykova, NV;Rampitsch, C;Ens, W
采用MALDI串联质谱技术，结合双向凝胶电泳、蛋白酶消化和液相色谱技术，对番茄中一种新的糖蛋白--病程相关的枯草杆菌蛋白酶样蛋白酶P69 B进行了糖基化鉴定和结构表征。从MS中通过其在糖型之间的特定m/z间距模式（203、162、146 u）鉴定来自微柱反相HPLC沉积在MALDI靶标上的糖肽级分。在大多数情况下，糖肽的[M + H]（+）离子的MS/MS光谱特征峰可用于确定糖组成、肽序列，从而确定可能的糖基化位点。此外，肽相关的产物离子可以很容易地用于数据库搜索程序，以确定糖蛋白。预测的5个糖基化位点中有4个具有生物学相关性，每个位点被5个N-连接聚糖侧链占据。此外，片段化效率允许检测含甲硫氨酸糖型的进一步修饰，无论是氧化或碘乙酰胺烷基化甲硫氨酸。通过MALDI-Qq-TOF提供的高分辨率允许从有限数量的材料中快速和灵敏地对位点特异性N-糖基化进行结构表征，并揭示了不同水平的异质性，包括不同的聚糖侧链修饰和相同糖基化位点上寡糖结构的异质性。
MALDI tandem mass spectrometry analysis on a hybrid quadrupole-quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a novel glycoprotein, pathogenesis-related subtilisin-like proteinase P69B from tomato. Glycopeptide fractions from microcolumn reversed-phase HPLC deposited on MALDI targets were identified from MS by their specific m/z spacing patterns (203, 162, 146 u) between glycoforms. In most cases, MS/MS spectra of [M + H](+) ions of glycopeptides featured peaks useful for determining sugar compositions, peptide sequences, and thus probable glycosylation sites. Furthermore, peptide-related product ions could readily be used in database search procedures to identify the glycoprotein. Four out of five predicted glycosylation sites were biologically relevant and occupied by five N-linked glycan side chains each. In addition, the fragmentation efficiency allowed detection of further modification of methionine-containing glycoforms, with either oxidized or iodoacetamide alkylated methionine. The high resolution furnished by MALDI-Qq-TOF allowed rapid and sensitive structural characterization of site-specific N-glycosylation from a limited quantity of material and revealed heterogeneity at different levels, including different glycan side-chain modifications, and heterogeneity of oligosaccharide structures on the same glycosylation site.