离子迁移谱-质谱技术及其在蛋白质糖基化研究中的应用

糖基化是生物体内重要的蛋白质翻译后修饰（PTM）。基于质谱的检测技术,特别是液相色谱-质谱（LC-MS）联用技术是糖基化研究的常规手段。然而,由于糖基化存在复杂的异构现象,大多数基于质谱的分析技术在区分其异构体时存在困难。离子迁移谱（IM）是一种基于离子在气相中的形状和电荷进行分离的技术,能够为质谱分析提供新的分离维度。离子迁移谱-质谱（IM-MS）技术具有较强的区分异构体的能力,在蛋白质糖基化研究中的潜力越来越受到重视。本文总结了IM-MS技术的类型和基本原理,及其在蛋白质糖基化研究中的进展,如对完整糖蛋白、完整糖肽和聚糖的研究,并展望了其应用前景。     

应用基于非变性质谱及串联质谱的碎片互补法分析完整IgA2抗体及其尺寸变异体

在完整蛋白及其复合物层面表征糖蛋白可提供修饰程度、变异体分布、结合计量关系等方面的重要结构信息。大量天然蛋白质及蛋白质药物都以糖蛋白形式存在,随着糖基化程度的增大,蛋白体系的异质性增强,糖链与肽链理化性质的差异使得常规表征手段容易损失准确性。使用质谱测定会因蛋白型质量分布展宽造成信号重叠,以及不同糖型离子化响应差异造成不同价态蛋白型质量分布差异而损失准确度,甚至无法实现分子质量测定。为解决这一问题,开发了基于有限价态还原的非变性质谱方法,但这些方法有的存在还原效率有限,有的需使用仅个别型号商品化仪器才具备的基于电子/质子转移的气相反应功能,应用范围受到限制。本工作针对含有非共价亚基的完整IgA2单克隆抗体及其尺寸变异体复合物体系,使用基于非变性质谱及串联质谱的碎片互补方法,以及碰撞诱导解离(CID)或高能碰撞解离(HCD)反应,综合利用有限价态还原效应和解离反应的质量守恒约束条件,实现高异质性糖蛋白复合物的测定。通过与针对全体蛋白型系综还原方法及蛋白型亚群有限还原方法的平行比较和交叉验证,评价各种方法的性能。结果表明,在解离产物特异性高且分子质量均一的情况下,碎片互补法可充分保证价态及...     

质谱蛋白全谱分析和指纹分析

质谱蛋白全谱分析和指纹分析可用于鉴别蛋白。应用酶或化学切解、修饰及序列反应,并与液相色谱或串级质谱联用,可给出进一步的结构信息,从而测定蛋白的组分、蛋白一级结构、表征序列、测定蛋白修饰位、基因变异位及缺失位,这两种方法可用于医疗诊断、蛋白的功能分析和蛋白组学研究中     

甲胎蛋白纯品的同位素稀释质谱法测定

建立了基于特征肽段的甲胎蛋白的液相色谱-同位素稀释串联质谱检测方法。选取3条同位素标记的甲胎蛋白特征肽段作为内标,准确称其质量后与酶切后的甲胎蛋白样品定量混合,采取Phenomenex Kinetex 2.6 μm C18色谱柱分离,电喷雾三重四极杆串联质谱多反应监测模式（MRM）测定,并对最优酶切条件、酶切效率以及定值结果的不确定度进行了考察和评定,得到的甲胎蛋白标准物质的最终测量结果为（0.329±0.016）mg/g。     

用尺寸排阻色谱测定蛋白质的构象转化点

建立了用尺寸排阻色谱研究蛋白质构象改变转化点的方法,测定了核糖核酸酶A在尺寸排阻色谱系统中的分配系数Kd,并用荧光检测器监测了加热所导致的蛋白质内源性荧光变化,从而计算出了核糖核酸酶A的热变性转化温度.通过液相色谱梯度洗脱系统在线增加尿素浓度,测定Kd值的动态变化,计算出使溶菌酶发生变性的尿素溶液浓度.用所建立的方法绘制了蛋白质变性前后的尺寸排阻色谱工作曲线,探讨了溶液pH对核糖核酸酶A热稳定性的影响.     

人尿液蛋白质N-糖肽富集和质谱鉴定的新方法研究

蛋白质的N-糖基化修饰参与了许多重要的生理过程,是研究多种重大疾病诊断标志物的热点。与其他体液活检样本相比,尿液可以无创、大量获取,并且不受稳态调节的影响,能够在一定程度上反映整个机体的生理和病理状态。因此,人尿液蛋白质N-糖基化的规模化研究对疾病诊断标记物的筛选和治疗靶点的发现均具有重大意义。由于人尿液中的N-糖蛋白含量有限,修饰比例较低,在质谱分析时N-糖肽易被高丰度的非糖肽所掩盖,难以鉴定。因此,发展高效、高选择性的富集材料是实现尿蛋白N-糖基化深度覆盖的必要前提。本研究利用巯基-烯点击化学反应,使用［3-(甲基丙烯酰氨基)丙基］二甲基(3-硫代丙基)氢氧化铵内盐（SPP）制备了两性离子修饰亲水硅胶材料（SPP-SiO₂）。该材料成功地应用于标准糖蛋白和健康人尿蛋白N-糖肽的富集和质谱检测,共鉴定了包含1 065个位点的633个尿液糖蛋白,比文献报道的鉴定规模提高了12.2%,证明了该亲水材料在尿液糖蛋白质组研究中的应用潜力。     

人血清中C反应蛋白的磁珠富集与质谱检测

采用免疫磁珠富集法,通过结合C反应蛋白单克隆抗体磁珠将血清中C反应蛋白提纯浓缩,用蛋白酶切质谱法对提纯物进行测定。同时对C反应蛋白酶切过程进行优化,提高酶切后肽图覆盖率至86%,选取特征肽段为ESDTSYVSLK。通过质谱定量特征肽段的浓度,得到血清中C反应蛋白的浓度为2.06μg/g。本实验建立血清中C反应蛋白的定量方法,血清中C反应蛋白的质谱定量测定奠定了基础。     

基质辅助激光解吸附飞行时间串联质谱法测定rhEPO唾液酸含量及鉴定糖基化位点

促红细胞生成素(EPO)是一种糖蛋白,绝大部分由肾脏产生,少量来自肝脏(胎儿及新生儿则主要由肝脏产生)[1].其最主要的功能是促进红细胞的生成,提高机体血红蛋白的浓度,改善机体的携氧能力,从而增强人体的耐力.EPO的肽链由165个氨基酸构成,分子量约为30～39 kDa,具有3个N-连接糖基化位点(Asn24、38、83)和1个O-连接糖基化位点(Ser126),平均含糖量约为40%[2].研究表明,重组EPO(rhEPO)糖基化程度对EPO的体内活性有相当大的影响,rhEPO比内源性EPO含有更多的三天线及四天线低聚糖,并且二者在唾液酸含量上有明显的差别[3,4].本工作采用基质辅助激光解吸-飞行时间串联质谱(MALDI-TOF-TOF-MS)测定重组EPO唾液酸含量,鉴定了三个N-连接糖基化位点,拟为重组EPO产品的质量控制以及与内源性EPO的区分提供新的思路及手段.     

糖蛋白的质谱分析策略

糖蛋白是一类由糖类和蛋白质以共价键连接而成的结合蛋白.它广泛存在于动物、植物、微生物及病毒中.近20年来的研究发现,糖蛋白中的糖基化修饰在各种生命现象中起着重要的作用.自19世纪末人们开始提出糖蛋白的概念并加以研究以来,先后明确了糖蛋白的糖基结构、多糖成分和部分生物学功能.同时,也能够识别糖蛋白中的糖苷键类型[1].     

应用非变性质谱-行波式离子淌度谱表征完整糖蛋白复合物的稳定性及小规模结构差异

在完整复合物层面表征糖蛋白复合物的高级结构和动态学,对于研究蛋白质功能具有重要意义。行波式离子淌度与非变性质谱结合不仅可提供额外的正交分离维度,还可通过测定碰撞截面积(CCS)获取分析物的几何特征信息,助力结构表征。本工作针对糖蛋白复合物稳定性及小规模结构差异的表征需求,选取由不同物种表达的亲和素作为糖蛋白模式复合物,探究离子淌度在非变性质谱分析中可提供的结构信息。结果表明,离子淌度可在完整复合物水平上分离蛋白型亚群分布,能够反映高阶复合物的几何特征,以及蛋白在气相解离条件、电荷调控条件和经电荷调控的气相解离条件下的构象变化程度,可应用于糖蛋白复合物体系的稳定性评价。     

Simulation of combustion in a porous-medium diesel engine

The future internal-combustion (IC) engines should have minimum emissions level under lowest feasible fuel consumption. This aim can be achievable with a homogeneous combustion process in diesel engines. We used a porous medium (PM) to homogenize the combustion process. This research studies simulation of a direct-injection diesel engine, equipped with a chemically inert hemispherical PM. Methane is injected into a hot PM, assuming mounted up the cylinder in head. Combustion with lean mixture occurs inside PM. A numerical model of PM engine was carried out using a modified version of the KIVA-3V code. PM results were evaluated with experimental data of unsteady combustion-wave of methane in a porous tube. The results show the mass fraction of methane, CO, NO and temperature in solid and gas phases of the PM and in-cylinder fluid. Also presented are the effects of injection timing and compression ratio on combustion.

     

Magnetic domain imaging of nano‐magnetic films using magnetic force microscopy with polar and longitudinally magnetized tips

Perpendicular or parallel magnetic fields are used to magnetize the tips used in magnetic force microscopy (MFM). In this process, perpendicular or parallel magnetic dipole moments are produced on the tip plane, thus leading to the formation of polar magnetized tips (PM‐tips) or longitudinally magnetized tips (LM‐tips), respectively. The resolution of an MFM image of a magneto‐optic disk is used for calibration of these tips, and the saturated magnetic fields of the PM‐ and LM‐tips are found to be 2720 Oe and 680 Oe, respectively. Because both tips can simultaneously magnetize the sample during the scanning process when measuring a Co thin film, clear MFM images are captured, which enable the identification of magnetizable regions and the distribution of the magnetic domains on the sample surface. These results will be useful for improving the manufacturing processes required for soft nano‐magnetic film production.     

基于缺陷分布形式的粉末冶金涡轮盘可靠度计算模型

为研究微缺陷对粉末冶金涡轮盘可靠性的影响,在有限元分析结果的基础上,基于国内粉末盘中缺陷分布的概率特 点,建立针对低循环疲劳裂纹萌生和裂纹扩展失效模式的可靠度计算模型。该模型可计算不同缺陷分布形式、不同循环载荷条件下的轮盘可靠度。对某型粉末盘失效概率的分析表明：表面质量是导致粉末盘失效的重要因素,这同大量试验研究结果相一致,对粉末盘的设计和使用具有工程指导意义。     

ICP—MS测定PM2．5中重金属不同消解方法的比对研究

采用电热板消解、微波消解和全自动石墨消解3种方法对大气PM2.5中的6种重金属用ICP-MS进行测定。全自动石墨消解法的加标回收率为94％～108％,RSD为1．5％～3．4％。实验证明该方法提高了样品前处理自动化程度,提高了操作规范化程度,减少待测物的损失,提高了方法的精密度和准确度,前处理简单,减轻了分析人员劳动强度,提高了工作效率,能更好地适应当前日益繁重的监测任务。     

Tillandsia stricta Sol (Bromeliaceae) leaves as monitors of airborne particulate matter—A comparative SEM methods evaluation: Unveiling an accurate and odd HP‐SEM method

Airborne particulate matter (PM) has been included among the most important air pollutants by governmental environment agencies and academy researchers. The use of terrestrial plants for monitoring PM has been widely accepted, particularly when it is coupled with SEM/EDS. Herein, Tillandsia stricta leaves were used as monitors of PM, focusing on a comparative evaluation of Environmental SEM (ESEM) and High‐Pressure SEM (HPSEM). In addition, specimens air‐dried at formaldehyde atmosphere (AD/FA) were introduced as an SEM procedure. Hydrated specimen observation by ESEM was the best way to get information from T. stricta leaves. If any artifacts were introduced by AD/FA, they were indiscernible from those caused by CPD. Leaf anatomy was always well preserved. PM density was determined on adaxial and abaxial leaf epidermis for each of the SEM proceedings. When compared with ESEM, particle extraction varied from 0 to 20% in air‐dried leaves while 23–78% of particles deposited on leaves surfaces were extracted by CPD procedures. ESEM was obviously the best choice over other methods but morphological artifacts increased in function of operation time while HPSEM operation time was without limit. AD/FA avoided the shrinkage observed in the air‐dried leaves and particle extraction was low when compared with CPD. Structural and particle density results suggest AD/FA as an important methodological approach to air pollution biomonitoring that can be widely used in all electron microscopy labs. Otherwise, previous PM assessments using terrestrial plants as biomonitors and performed by conventional SEM could have underestimated airborne particulate matter concentration.     

考虑机会维修的等风险预防性维修策略优化

研究了对流水线生产系统进行等风险预防性维修策略优化的问题。预防性维修计划中包括不完全预防性维修和预防性替换，突发故障用最小维修处理。为了减少预防性维修造成的停产损失，一些预防性维修活动将根据机会维修阈值归并在一起进行。采用遗传算法在满足系统可靠性的前提下，以最小化维修成本为目标优化预防性维修计划。首先建立预防性维修的优化模型；然后设计了模型求解的遗传算法；最后在Emplant仿真环境下，将算法求解的最优预防性维修计划应用到生产系统仿真模型中进行评价，并与传统的故障替换策略进行了比较。     

Recent advancements in glycoproteomic studies: Glycopeptide enrichment and derivatization,characterization of glycosylation in SARS CoV2, and interacting glycoproteins

Proteomics studies allow for the determination of the identity, amount, and interactions of proteins under specific conditions that allow the biological state of an organism to ultimately change. These conditions can be either beneficial or detrimental. Diseases are due to detrimental changes caused by either protein overexpression or underexpression caused by as a result of a mutation or posttranslational modifications (PTM), among other factors. Identification of disease biomarkers through proteomics can be potentially used as clinical information for diagnostics. Common biomarkers to look for include PTM. For example, aberrant glycosylation of proteins is a common marker and will be a focus of interest in this review. A common way to analyze glycoproteins is by glycoproteomics involving mass spectrometry. Due to factors such as micro- and macroheterogeneity which result in a lower abundance of each version of a glycoprotein, it is difficult to obtain meaningful results unless rigorous sample preparation procedures are in place. Microheterogeneity represents the diversity of glycans at a single site, whereas macroheterogeneity depicts glycosylation levels at each site of a protein. Enrichment and derivatization of glycopeptides help to overcome these limitations. Over the time range of 2016 to 2020, several methods have been proposed in the literature and have contributed to drastically improve the outcome of glycosylation analysis, as presented in the sampling surveyed in this review. As a current topic in 2020, glycoproteins carried by pathogens can also cause disease and this is seen with SARS CoV2, causing the COVID-19 pandemic. This review will discuss glycoproteomic studies of the spike glycoprotein and interacting proteins such as the ACE2 receptor.     

积雪草苷和羟基积雪草苷的电喷雾质谱裂解机制研究

以积雪草苷和羟基积雪草苷为研究对象,在正负离子模式下,获得其电喷雾离子阱三级质谱图,结合分子模拟方法,解析了其正负离子模式下二级质谱中基峰子离子的三级质谱裂解途径,并寻找其裂解规律。结果表明,以上两种皂苷化合物在正负离子模式下,分子离子均发生苷键的断裂,主要生成苷糖基的碎片离子;苷糖基在三级质谱裂解中主要发生脱水和交叉环切除的裂解反应,其质谱裂解途径应遵循苷糖基的脱水和缩环时不破坏分子内氢键的裂解规律。应用密度泛函(DFT)方法优化得到苷糖基能量最低的稳定构型,利用氢键识别模块对苷糖基可能形成的分子内氢键及其位置进行了预测。实验结果表明,推测的正负碎片离子与质谱裂解结果相符,从而验证了以上苷糖基的脱水和缩环规律的合理性。本实验结果丰富了乌苏烷型皂苷的电喷雾质谱裂解规律,可为乌苏烷型皂苷的结构鉴定提供更多支持。     

荧光分光光度法测定滴眼液中萘甲唑林的含量

目的 :荧光分光光度法测定滴眼液中萘甲唑林的含量。方法 :激发波长2 81nm ,发射波长 32 7nm ,狭缝 5nm ;光电倍增管增益 :低 ;时间扫描速度 10nm/min。结果 :萘甲唑林在 10 0～ 5 0 0ng/mL范围内线性关系良好。回归方程Y =2 38+175 7X　r=0 9998。萘甲唑林滴眼液及淡泪净滴眼液的 3种浓度平均回收率 (n =9)分别为10 0 36 % ,10 0 0 3% ;RSD分别为 0 2 6 % ,0 2 7%。结论 :本法专属性强 ,结果准确。可用于滴眼液中萘甲唑林的含量测定。     

移动型3-UPU并联机构静态稳定性分析

使用最小势能原理,对刚性支撑3-UPU并联机构进行了静态稳定性分析。稳定性分析中,将并联机构的连杆分解为包含油液在内的三部分,通过建立并联机构的Lagrange势能函数,得到反映机构静态稳定性的Hessian矩阵,并由此提出了机构不同稳定性状态的比较判据。分析和计算表明,在工作空间中,移动型3-UPU并联机构在由几何构型所确定的工作空间中的静平衡基本上稳定。