Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Glycobiology. 2013 Feb;23(2):211-21. doi: 10.1093/glycob/cws144. Epub 2012 Oct 15.
In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of "robust enrichment" afforded by covalent-labeling techniques and "specificity for glycoproteins" typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status.
在本文中，我们提出了两种互补的策略来富集活细胞中的糖蛋白，这两种策略结合了共价标记技术提供的“丰富富集”的理想属性和凝集素或抗体亲和试剂通常提供的“糖蛋白特异性”。我们的策略涉及选择性地将醛基引入唾液酸（通过过碘酸盐氧化（过碘酸盐氧化和苯胺催化肟连接（PAL）））或末端半乳糖和 N-乙酰半乳糖胺残基（通过半乳糖氧化酶（半乳糖氧化酶和苯胺催化肟连接（GAL））），然后用氨基氧基生物素进行苯胺催化肟连接，以高效且保持细胞活力地生物素化糖蛋白亚群的聚糖。正如预期的那样，与唾液酸缺乏的细胞相比，当应用于完全唾液酸化的细胞时，这两种方法表现出相互的标记效率。为了评估这些标记方法在糖蛋白质组学中的应用，我们通过吸附到固定化链霉亲和素上来富集 PAL 和 GAL 标记（生物素化）的糖蛋白组。然后，通过液相色谱-串联质谱法对总胰蛋白酶肽和随后从仍结合到珠子上的 N-聚糖释放的肽进行分析，获得糖蛋白身份（ID）和 N-糖基化位点信息使用肽 N-糖苷酶 F。总共鉴定出 175 个独特的 N-糖基化位点，属于 108 个非冗余糖蛋白。在 108 个糖蛋白中，有 48 个是通过两种标记方法鉴定的，其余的是通过在唾液酸化的细胞上使用 PAL（40 个）或在唾液酸缺乏的细胞上使用 GAL（20 个）鉴定的。我们的结果表明，PAL 和 GAL 可以作为糖蛋白亚群的靶向蛋白质组学的化学标记的互补方法，以及鉴定具有改变的唾液酸化状态的细胞中蛋白质的糖基化位点。
