蛋白质分离和翻译后修饰肽段富集新方法的研究

本论文发展了新型蛋白质分离混合模式离子交换色谱法，以及针对磷酸肽和N-连接糖 肽的选择性富集技术，并将其成功地应用于复杂样品蛋白质或肽段混合物的分离富集中。 研制了分别以聚合物整体材料和无孔聚合物微球为基质的弱阴-弱阳混合模式离子交 换色谱微柱。在与胰蛋白酶酶解兼容的条件下(pH8)，实现了酸性、碱性以及中性蛋白质 的同时分离；获得了比单一模式的离子交换色谱更高的分辨率。 以有机-无机杂化硅胶整体材料作为基质，采用戊二醛偶联法固载了磷酸螯合基团，进 而制备了一种新型Ti4+-IMAC 整体材料；并将其用于鼠肝线粒体提取蛋白质的分析。结果 表明：该材料具有选择性好、富集容量高、回收率高以及重现性好等优点。 分别研制了以Fe3O4 磁性纳米颗粒为载体的胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶反应 器，并将多酶酶解策略与杂化硅胶基质Ti4+-IMAC 整体柱的富集方法相结合，从人神经母 细胞瘤细胞的膜组分中鉴定到43 个磷酸化蛋白质，其中14 个定位于膜上，9 个含跨膜区。 发展了聚乙二醇偶联修饰和超滤分离相结合的方法，实现了N-连接糖肽的选择性富 集。该富集效果同商品化酰肼树脂微球富集效果相当，但是操作更为简便、选择性更好、 抗干扰能力更强

Combining mPEG-Hz Derivatization and Filter-Based Size Exclusion for Selective Enrichment of N-glycopeptides

Although hydrazide (Hz) is widely used for the selective enrichment of N-glycopeptides, it takes a long time to wash off the non-specifically adsorbed peptides on the Hz beads. To solve this problem, in our recent work, a strategy with the combination of methoxy-polyethylene glycol (mPEG)-Hz based derivatization and filter-based size exclusion, was developed, and successfully applied for the facile enrichment of N-glycopeptides. Herein, glycoproteins were first digested, followed by the oxidization of the cis-diol groups of carbohydrates on glycopeptides to aldehydes, which could further form covalent hydrazone bonds with mPEG-Hz. With the increased molecular weights, the derivatized glycopeptides could be separated from other peptides by a centrifugal filter device. After that, PNGase F was added to cut off mPEG-conjugated carbohydrates, which could be kept in the retentate, while the released peptides could be collected in filtrate after centrifuge. HRP, a standard glycoprotein, was used to evaluate this protocol. With 10-kDa mPEG-Hz for N-glycopeptide enrichment, and filter devices with nominal molecular weight limit of 10 kDa to removal non-glycopeptides, 5 peptides with N-glycosite were detected, with the specificity consistent to that obtained with the commercial hydrazide beads. Most importantly, the removal of non-glycopeptides became easy by filter-based size exclusion, beneficial to shorten the sample preparation time. With the further optimization of the size of mPEG-Hz and the cut off molecular weight of filter, improved selectivity of glycopeptide enrichment is expected to achieve, which might be promising in N-glycoproteome analysis.Although hydrazide (Hz) is widely used for the selective enrichment of N-glycopeptides, it takes a long time to wash off the non-specifically adsorbed peptides on the Hz beads. To solve this problem, in our recent work, a strategy with the combination of methoxy-polyethylene glycol (mPEG)-Hz based derivatization and filter-based size exclusion, was developed, and successfully applied for the facile enrichment of N-glycopeptides. Herein, glycoproteins were first digested, followed by the oxidization of the cis-diol groups of carbohydrates on glycopeptides to aldehydes, which could further form covalent hydrazone bonds with mPEG-Hz. With the increased molecular weights, the derivatized glycopeptides could be separated from other peptides by a centrifugal filter device. After that, PNGase F was added to cut off mPEG-conjugated carbohydrates, which could be kept in the retentate, while the released peptides could be collected in filtrate after centrifuge. HRP, a standard glycoprotein, was used to evaluate this protocol. With 10-kDa mPEG-Hz for N-glycopeptide enrichment, and filter devices with nominal molecular weight limit of 10 kDa to removal non-glycopeptides, 5 peptides with N-glycosite were detected, with the specificity consistent to that obtained with the commercial hydrazide beads. Most importantly, the removal of non-glycopeptides became easy by filter-based size exclusion, beneficial to shorten the sample preparation time. With the further optimization of the size of mPEG-Hz and the cut off molecular weight of filter, improved selectivity of glycopeptide enrichment is expected to achieve, which might be promising in N-glycoproteome analysis

人神经母细胞瘤的磷酸化膜蛋白质组分析

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱（Ti 4＋-IMAC）整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/LNaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti 4＋-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选

一种固定化金属离子亲和色谱整体柱的制备方法

本发明涉及固定化金属离子亲和色谱材料的制备，具体地说是一种固定化金属离子亲和色谱整体柱的制备方法，以表面带有醛基的有机-无机杂化硅胶整体材料为载体制成整体柱材料；于整体柱材料上连续通过含有胺甲基磷酸和氰基硼氢化钠NaCNBH3的磷酸盐缓冲溶液，制备得到表面带有磷酸螯合基团的整体材料载体；将过渡金属离子的水溶液通过材料载体，制备得到固定化金属离子亲和色谱整体柱。利用该固定化金属离子亲和色谱整体柱可以实现混合肽段中磷酸化肽的高选择性富集。本发明的优点为：对磷酸化肽具有较好的选择性，可反复使用，富集容量较大，回收率较高，且制备方法简便，通透性好、反压小、机械强度好、耐酸碱性强。待填

Analysis on Wind Resistance Index of Multi-rotor UAV

Since multi-rotor UAV possesses simple structure and light weight, it is likely to be affected by the outside wind field. So far, researches on the wind resistance of multi-rotor UAV mostly focus on the control method of anti-interference. However, a common index to measure the ability of wind resistance is still absent. For this reason, the article does quantitative analysis on the induced velocity of propellers and propeller thrust of a multi-rotor UAV. What's more, the article analyzes the influence of horizontal wind on the drone and builds a model on a multi-rotor UAV in the wind field. Finally, the article puts forward a parameter to measure the wind resistance ability of a multi-rotor UAV