Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry
Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry
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DOI：
10.1021/pr0502065
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发表时间：
2005-11-01
影响因子：
4.4
通讯作者：
Smith, RD
中科院分区：
文献类型：
--
作者：
Liu, T;Qian, WJ;Smith, RD
人类血浆蛋白质组的巨大复杂性、相对蛋白质丰度的宽动态范围(超过10个数量级)和巨大的异质性(由于翻译后修饰，如糖基化)严重挑战了现有分析方法的能力。在这里，我们描述了一种使用免疫亲和减法和糖蛋白捕获相结合的方法来广泛分析人血浆N-糖蛋白的方法，以降低蛋白质浓度范围和总体样本复杂性。使用预填充的固定化抗体柱同时去除六种高丰度血浆蛋白。然后用肼树脂从耗尽的血浆中捕获N-连接的糖蛋白，并用N-糖苷酶F(PNGase F)释放结合的N-连接的糖肽。在强阳离子交换(SCX)分离后，用反相毛细管液相色谱-串联质谱仪(LC-MS/MS)分析脱糖多肽。使用严格的标准，总共有2053个不同的N-糖肽被自信地鉴定出来，覆盖了303个非冗余N-糖蛋白。这种浓缩策略显著提高了对低丰度蛋白质的检测，并使其能够识别出一些低丰度蛋白质，例如白细胞介素1受体拮抗剂蛋白(类似于200pg/mL)、组织蛋白酶L(类似于1 ng/mL)和转化生长因子β1(类似于2 ng/mL)。共鉴定了639个N-糖基化位点，并初步证明了通过高分辨率液相色谱与傅里叶变换离子回旋共振质谱仪(LC-FTICR)相结合的准确质量测量来评估这些糖基化位点的总体高精度。
The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (similar to 200 pg/mL), cathepsin L (similar to 1 ng/mL), and transforming growth factor beta 1 (similar to 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.