Liu Tao, Qian Wei-Jun, Gritsenko Marina A, Camp David G, Monroe Matthew E, Moore Ronald J, Smith Richard D
Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, USA.
J Proteome Res. 2005 Nov-Dec;4(6):2070-80. doi: 10.1021/pr0502065.
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 (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 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.
人血浆蛋白质组具有极大的复杂性、感兴趣的相对蛋白质丰度的宽动态范围（超过10个数量级）以及巨大的异质性（由于翻译后修饰，如糖基化），这对现有分析方法的能力提出了严峻挑战。在此，我们描述了一种使用免疫亲和扣除和糖蛋白捕获相结合的方法对人血浆N-糖蛋白进行广泛分析，以降低蛋白质浓度范围和整体样品复杂性。使用预装的固定化抗体柱同时去除六种高丰度血浆蛋白。然后使用酰肼树脂从耗尽的血浆中捕获N-连接糖蛋白并进行酶切，使用肽-N-糖苷酶F（PNGase F）释放结合的N-连接糖肽。经过强阳离子交换（SCX）分级分离后，对去糖基化肽进行反相毛细管液相色谱-串联质谱（LC-MS/MS）分析。使用严格的标准，共可靠鉴定出2053种不同的N-糖肽，覆盖303种非冗余N-糖蛋白。这种富集策略显著提高了检测能力，并能够鉴定出许多低丰度蛋白，如白细胞介素-1受体拮抗剂蛋白（约200 pg/mL）、组织蛋白酶L（约1 ng/mL）和转化生长因子β1（约2 ng/mL）。共鉴定出639个N-糖基化位点，并初步证明了通过使用高分辨率液相色谱-傅里叶变换离子回旋共振质谱（LC-FTICR）进行精确质量测量评估的这些糖基化位点分配的总体高精度。
