毛细管电泳-质谱联用技术研究进展

毛细管电泳-质谱（CE-MS）联用技术兼具毛细管电泳高效分离能力与质谱高灵敏检测、高真度定性的优势,已成为物质分离分析研究的一种非常重要的工具。本文对近几年来 CE-MS 联用的关键技术及 CE-MS 在中药分析、环境检测等领域的一些应用进展进行综述,对其发展进行了展望。     

全柱成像毛细管等电聚焦电泳测定艾塞那肽等电点

目的:采用新一代全柱成像毛细管等电聚焦电泳技术(CIEF-WCID)测定艾塞那肽等电点。方法:采用互补性金属氧化物半导体成像技术对样品等电聚焦过程进行实时记录,根据适宜的marker计算得到艾塞那肽的等电点,并对方法的准确度与重复性进行考察。结果:测得艾塞那肽等电点为5.46,与凝胶电泳结果基本一致,相对标准偏差为0.11%。CIEF-WCID方法快速准确,相对误差小于2.5%,重复性良好。结论:CIEF-WCID作为一种新的技术手段可用于艾塞那肽等电点的分析,方法快速、准确、重复性好,可为多肽的质量控制提供一种可靠的分析方法。     

用固相pH梯度等电聚焦分析Gc蛋白亚型

用固相pH梯度等电聚焦技术及免疫固定技术对201名北京地区无关汉族群体的人血清Gc蛋白亚型进行了分型鉴定及基因频率的调查.Gc^1F为0.3698.Gc^1S为0.2812.Gc²为0.3490.观察值与期望值吻合良好(Σx²=1.057,P＞0.70)     

电喷雾萃取电离技术在蛋白质分析中的应用及展望

电喷雾萃取电离技术(extractive electrospray ionization,EESI)是一种能灵敏地电离固体、液体、气体、黏性样品等复杂基体中痕量大分子和小分子的新兴软电离技术．在简要介绍EESI原理的基础上,着重综述其在蛋白质分析中的应用．与商品化质谱仪器配置的电喷雾电离源(electrospray ionization,ESI)不同,EESI能够在常压条件下最大程度地保留蛋白质在样品中的原始构象,并获得大量具有生物活性的蛋白质离子．由此可见,EESI及类似的技术在蛋白质芯片制备、高分辨率氢/氘交换质谱(蛋白质结构分析)、蛋白质计量等方面具有良好的应用前景．本文也对其发展趋势进行了展望．     

纳升电喷雾串联质谱技术对阻断泛素通路诱导凋亡相关蛋白质的鉴定

应用毛细管液相色谱 电喷雾 四极杆 飞行时间串联质谱和纳升电喷雾 四极杆 飞行时间串联质谱技术 ,对阻断白血病细胞泛素通路诱发的凋亡相关蛋白质进行了鉴定。通过双向电泳发现 ,蛋白质斑点H在阻断Mo7e白血病细胞泛素通路 2h之后 ,表达量明显增加 ,6h达到最高。该斑点经MALDI TOF MS肽质量指纹谱分析未获结果 ,但通过上述 2种串联质谱技术获得其胰蛋白酶水解肽段的串联质谱图和肽段的全长序列 ,经检索均确认为RhoGDIβ蛋白。进一步发现阻断泛素通路还诱发了另 2个斑点出现 ,位于H点附近 ,经鉴定为同一蛋白质 ,可能是不同翻译后修饰所造成     

目前最高分辨率的电泳──固相pH梯度等电聚焦

固相pH梯度等电聚焦是国际上80年代的新型电泳技术.利用一系列具有弱酸和弱碱性质的丙烯酰胺衍生物滴定时,在滴定终点附近形成的pH梯度并参与丙烯酰胺的共价聚合,从而形成固定的不随环境电场等条件变化的pH梯度,该方法具有比传统载体两性电解质等电聚焦更高的分辨率、更大的上样量.可用于分析和制备相近pI的蛋白质,多肽等.     

等电聚焦法测定2.5S NGF的等电点

采用水平等电聚焦法测定了２５Ｓｍ神经生长因子（ＮＧＦ）的等电点。经测定,纯化的２５ＳｍＮＧＦ的等电聚焦为三条带,这与文献报道相一致,等电点ｐＨ值分别为ｐＨ８７、９０、９３,其主要成分为β亚基ＮＧＦ及其修饰物的混合物,包括在羧基端失去８个氨基酸残基和在氨基端失去１个精氨酸残基,由这三种组分构成的ＮＧＦ统称为２５ＳｍＮＧＦ。     

循环自由流等电聚焦方法的建立及应用研究

等电聚焦是利用蛋白质分子或其它两性电解质分子的等电点不同,在一个连续、稳定、线性的pH梯度中进行蛋白质分析分离的技术。薄层凝胶等电聚焦具有高分辨率,但上样量小,聚焦后样品分析处理不便,仅局限于分析。循环自由流液相等电聚焦以液体循环流动代替固相载体,上样量大,聚焦后样品分析处理方便,有较大的制造潜力。Bier等已开展这项技术的研究。该技术应用到遗传工程上可对干扰素等进行精制纯化^[3]。本实验应用循环自由流等电聚焦仪(Recycling free-flow isoelectric fo-cusing,RIEF)进行了pH梯度的建立、模型化合物的分离、相关参数的比较,并被步应用于生物活性蛋白的精制和活性回收,旨在为遗传工程产品下游工艺提供一种新型而行之有效的方法。     

利用等电聚焦电泳研究黄单胞菌分类

对黄单胞菌的4个种及29个致病变种的代表菌株进行了等电聚焦电泳研究,发现所测定的黄单胞菌种间、致病变种间蛋白质图谱差别很大。电泳结果经聚类分析表明,某些致病变种间的差别并不一定比种间的差别小,说明某些致病变种可以上升到种这一分类地位。引起细菌性黑颖病的黄单胞菌禾谷致病变种、小麦致病变种、大麦致病变种间差别很小,仅有6条蛋白质谱带的差别。因此这3个致病变种的分类地位需重新考虑,同时说明等电聚焦电泳对黄单胞菌种下分类具有一定指导意义。     

多肽分子疏水性的电喷雾飞行时间质谱法快速测定

疏水作用是决定生物分子的结构和性质的重要因素,特别是在蛋白质的折叠,药物分子与受体(蛋白质、DNA等)的相互作用中起着关键作用.分子疏水性的强弱决定于分子内非极性基团的含量.在一定的实验条件下,电喷雾所获得的信号与多肽分子内非极性基团的面积呈现良好的相关性.因此,采用电喷雾飞行时间质谱法,在数分钟之内快速测定了不同多肽之间的疏水性,所获得结果与色谱法结果一致.     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

Sequencing of Gel-Isolated Proteins Using Microblotter Capillary Liquid Chromatography-Electrospray Mass Spectrometry

Enzymatic digests of proteins isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were separated by capillary high-performance liquid chromatography (HPLC). The column eluate was split to an electrospray mass spectrometer on one side and to both a UV detector and a microblotter on the other side. Using the microblotter, the peptides eluted from the column were collected directly onto a polyvinylidene difluoride (PVDF) membrane for Edman sequencing. Thus, a peptide mass map from the mass spectrometric analysis and a prepared PVDF membrane for subsequent Edman sequencing were generated in a single experiment. The addition of molecular mass information to the blotted LC eluate is useful for determining the most important peaks to undergo Edman sequencing. Coupling the capillary HPLC with a microblotter to electrospray mass spectrometry provides an integrated system for separation, collection, and structural analysis of protein digests. It provides high levels of sensitivity, recovery, and convenience for protein characterization. Proteins loaded onto SDS–PAGE at low picomole levels can be analyzed by the new integrated system.     

Combination of Micropreparative Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Peptide Analysis

Signal suppression is a problem in matrix-assisted laser desorption/ionization mass spectrometry of peptides prepared by capillary electrophoresis. Many common electrolytes that are efficient for separation, such as sodium phosphate, also are strongly suppressive during laser desorption/ionization. We have tested individual electrolytes for highest performance in each step of separation and collection, respectively. Suppression is not observed if citrate, trifluoroacetic acid, or hydrochloric acid is used for collection, while phosphate still can be employed in the capillary providing excellent resolution. Low concentrations of hydrochloric acid added to the sample/matrix mixture generate mass spectra with better ion intensities than if trifluoroacetic acid or citrate is used.     

Electrospray Ionization Mass Spectrometric Study of the Multiple Intracellular Monomeric and Polymeric Hemoglobins of Glycera dibranchiata

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dibranchiata is comprised of two groups of globins differing in their primary structures and state of aggregation. About six electrophoretically and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the usual distal His, have been identified to date. Deconvolution of the electrospray ionization mass spectra (ESI-MS) of the Hbs and of their carbamidomethylated, reduced, and reduced/carbamidomethylated forms, using a maximum entropy-based approach (MaxEnt), showed the presence of at least 18 peaks attributable to monomer Hbs (14,500–15,200 Da) and an approximately equal number of polymer Hb peaks (15,500–16,400 Da). Although the ratio of the monomer to polymer components in pooled Hb preparations remained constant at 60:40, Hb from individuals had generally less than 6 monomer and 6 polymer components; 2 of the 19 individuals appeared to be deficient in polymer Hbs. Taking into account possible fragmentations of the known monomeric and polymeric globin sequences, we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Surprisingly, the calculated mass of the sequence deduced from the high-resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 components, of which 5, accounting for 67% of total, were related to the three major sequences GMG2–4. These findings underline the need for routine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt processing illustrates the power of this method to resolve complex protein mixtures.     

一种SDS-PAGE与MALDI-TOF质谱联用的方法

以尿激酶原为材料,探索一种从SDS-PAGE胶上回收蛋白质做MALDI-TOF质谱的方法.所用的回收方法包括电洗脱、脱盐、除SDS等过程.电洗脱用的是高盐阻断法,脱盐用的是超滤技术,去除SDS用的是冷丙酮沉淀法.结果证明,此方法至少对一些蛋白质（如尿激酶原和牛血清白蛋白）是可行的.     

一种SDS-PAGE与MALDI-TOF质谱联用的方法

以尿激酶的为材料,探索一种从ＳＤＳ－ＰＡＧＥ胶上回收蛋白 做ＭＡＬＤＩ－ＴＯＦ质谱的方法,所用的回收方法包括电洗脱、脱盐、除ＳＤＳ等过程,电洗脱用的是高盐阻断法,脱盐用的超滤技术,去除ＳＤＳ用的是冷丙沉淀法,结果证明,此方法至少对一些蛋白质是可行的。     

Localized Sampling Enables Monitoring of Cell State via Inline Electrospray Ionization Mass Spectrometry

Nascent advanced therapies, including regenerative medicine and cell and gene therapies, rely on the production of cells in bioreactors that are highly heterogeneous in both space and time. Unfortunately, advanced therapies have failed to reach a wide patient population due to unreliable manufacturing processes that result in batch variability and cost prohibitive production. This can be attributed largely to a void in existing process analytical technologies (PATs) capable of characterizing the secreted critical quality attribute (CQA) biomolecules that correlate with the final product quality. The Dynamic Sampling Platform (DSP) is a PAT for cell bioreactor monitoring that can be coupled to a suite of sensor techniques to provide real-time feedback on spatial and temporal CQA content in situ. In this study, DSP is coupled with electrospray ionization mass spectrometry and direct-from-culture sampling to obtain measures of CQA content in bulk media and the cell microenvironment throughout the entire cell culture process (≈3 weeks). Post hoc analysis of this real-time data reveals that sampling from the microenvironment enables cell state monitoring (e.g., confluence, differentiation). These results demonstrate that an effective PAT should incorporate both spatial and temporal resolution to serve as an effective input for feedback control in biomanufacturing.     

Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue

The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr³⁰⁸ and Ser⁴⁷³ upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser⁴⁷³ and Thr³⁰⁸ phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser⁴⁷³ and Thr³⁰⁸ phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser⁴⁷³-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons.