The Use of Carrier Ampholyte-Free Isoelectric Focusing for Proteomic Analysis

Carrier ampholyte-free isoelectric focusing was applied for pre-concentration, purification and micropreparation of phycobiliproteins (C-phycocyanin, allophycocyanin, B-phycoerythrin) extracted from cyanobacteria Anabeana doliolum and from red microalga Porphyridium cruentum. The extraction of phycobiliproteins was carried out in deionized water. The sonication in the ultrasonic bath and liquid nitrogen freeze grind was used for extraction of proteins of interest. Pre-concentrated and pre-separated proteins were collected and analyzed via MALDI-TOF-TOF mass spectrometer after their proteolytic digestion via trypsin. Based on tandem mass spectrometric analysis, the C-phycocyanin, allophycocyanin and B-phycoerythrin were identified unambiguously.

     

Carrier Ampholyte Isoelectric Focusing Based Two-Dimensional Electrophoresis in Preliminary Screening of Differential Proteomics Analysis

Two-dimensional electrophoresis is a current method for separating complex protein mixtures of a given sample in different states. In this study an improved carrier ampholyte isoelectric focusing method has been evaluated for its capacity for preliminary screening of expressional proteomics subjects. In comparison with current carrier ampholyte isoelectric focusing, this method showed enough resolution power to display major expressional changes in proteomic samples and demonstrated it can be used as a substitution for the immobiline based isoelectric focusing method.     

用于蛋白质分析的毛细管等电聚焦-电喷雾质谱接口的改进

蛋白质组学对其分析技术提出了大规模、高通量的要求 [1] .传统的等电聚焦 ( p I) -分子量 ( MW)双向电泳技术 ( 2 D- Gel)尽管在蛋白质组学研究中占有重要地位 ,但其操作繁杂、工作周期长 .Pandey等[1] 将毛细管等电聚焦 ( CIEF)与电喷雾质谱 ( ESIMS)联用 ,使得 p I和 MW两维分离鉴定技术变得简单迅速 .但 CIEF- MS的接口操作需中断高压和将毛细管阴极端插入电喷雾管 ,故引起分析蛋白质的散焦和不重现 .本工作改进了 CIEF- MS接口 ,采用毛细管阴极端和电喷雾针一体化的电喷雾接口 ,无需中断高压 ,实现了 CIEF- MS的在线联…     

Preparative Isoelectric Focusing

Electrophoretic mobility has been used for years as a basis for separation and characterization of proteins. The resolving power of conventional electrophoresis is limited, and the characterization by mobility is not unique because of the dependence of the protein mobilities on the pH and ionic strength of the buffers.     

毛细管等电聚焦分离蛋白质

采用均匀设计法进行未涂壁石英毛细管等电聚焦研究,考察两性电解质、甲基纤维素、四甲基乙二胺和牛磺酸几种组分对等电聚焦的影响,确定出了比较合适的浓度范围。当中性蛋白质迁移通过检测窗后,在进样端施加一低气压,从而使酸性蛋白质得到很好的分离,适用于较宽的ｐＩ范围。方法的重复性好,为蛋白质分离鉴定提供一种有力的工具。     

Isoelectric Focusing with Immobilized pH Gradients

Abstract

The technique of isoelectric focusing (IEF) via immobilized pH gradients (IPG) was first announced to the scientific community at a meeting of the International Electrophoresis Society in Athens, April 1982, as the result of an intensive collaborative effort [1]. In five years, the technique has been extensively developed in three fundamental aspects: analytical, preparative and as a first dimension of two-dimensional (2-D) maps. The merits and, recently, the flaws of the IPG technique have been evaluated and recognized, so that at the present writing we feel it is ready for successful introduction in most life-science laboratories.     

毛细管等电聚焦电泳技术进展

介绍了毛细管等电聚焦电泳(CIEF)技术的发展和现状,特别对载体两性电解质、柱内表面处理和检测技术等方面的最新进展加以系统评述,并且对CIEF的定性能力、峰容量、重现性及其在生命科学方面的应用前景进行了讨论。引用文献59篇。     

Direct MALDI-MS Analysis of Proteins Isolated by Liquidphase Isoelectric Focusing Electrophoresis

A liquid-phase isoelectric focusing electrophoresis system(Rotofor) was used as the prefractionation tool for the sample preparation in the MALDI-MS analysis of a protein mixture. Each fraction collected was then directly subjected to MALDI-TOF-MS analysis. By this approach, we are able to resolve two types of hemoglobins, A and C, which cannot be successfully separated by means of the traditional SDS-PAGE method.     

Novel Methods for Controlling the Current During Isoelectric Focusing

While studying the effect of electrochemical reactions occurring at the electrodes on achievement of steady state in isoelectric focusing (IEF) we observed an abnormal increase of the current. Because the magnitude of the current determines the progress of IEF, knowledge gained from studies of its nature and generation may enable this effect to be controlled. We observed that addition of gelatin to the electrode solutions suppresses the magnitude of the current flowing through the system; this enables IEF to be performed under conditions closer to steady state, and steady state is achieved more quickly.     

Intercompany Study to Evaluate the Robustness of Capillary Isoelectric Focusing Technology for the Analysis of Monoclonal Antibodies

Interlaboratory comparisons are essential to bringing emerging technologies into biopharmaceutical industry practice and regulatory acceptance. As a result, an international team including 12 laboratories from 10 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of capillary isoelectric focusing (CIEF) to assess the charge heterogeneity of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charge isoforms of a representative monoclonal antibody (rMAb) sample using the same CIEF method. Statistical evaluation of the data was performed to determine within and between-laboratory consistencies and outlying information. The apparent pI data generated for each charge variant peak showed very good precision between laboratories with percentage of RSD values of ??0.5%. Similarly, the percentage of RSD for the rMAb charge variants percent peak area values are ??4.4% across different laboratories with different analysts using different lots of ampholytes and multiple instruments. Taken together, these results validate the appropriate use of CIEF in the biopharmaceutical industry in support of regulatory submissions.     

液相等电聚焦结合双向凝胶电泳分离碱性蛋白

在蛋白组学研究中, 经典的双向凝胶电泳法(2-DE)对碱性蛋白及低丰度蛋白的分离存在技术障碍, 但预分离技术的应用可弥补其缺陷. 液相等电聚焦可有效地分离富集复杂蛋白样品. 碱性胶条用于2-DE可极大地提高蛋白上样量和凝胶分辨率. 将上述两种技术相结合用于碱性蛋白质和低丰度蛋白质的分离鉴定, 可使碱端区域双向凝胶图谱质量显著提高, 蛋白点更清晰且点数增多, 质谱鉴定确信度提高, 碱性蛋白和低丰度蛋白质谱鉴定成功率提高, 对于蛋白组学研究具有一定的意义.     

A Chemical Proteomic Probe for the Mitochondrial Pyruvate Carrier Complex

Target engagement assays are crucial for establishing the mechanism‐of‐action of small molecules in living systems. Integral membrane transporters can present a challenging protein class for assessing cellular engagement by small molecules. The chemical proteomic discovery of alpha‐chloroacetamide (αCA) compounds that covalently modify cysteine‐54 (C54) of the MPC2 subunit of the mitochondrial pyruvate carrier (MPC) is presented. This finding is used to create an alkyne‐modified αCA, YY4‐yne, that serves as a cellular engagement probe for MPC2 in click chemistry‐enabled western blotting or global mass spectrometry‐based proteomic experiments. Studies with YY4‐yne revealed that UK‐5099, an alpha‐cyanocinnamate inhibitor of the MPC complex, engages MPC2 with remarkable selectivity in human cells. These findings support a model where UK‐5099 inhibits the MPC complex by binding to C54 of MPC2 in a covalent reversible manner that can be quantified in cells using the YY4‐yne probe.     

毛细管等电聚焦/加压毛细管电色谱二维分离体系在多肽分离中的应用

以毛细管等电聚焦(cIEF)为第一维分离模式,以反相加压毛细管电色谱(pCEC)为第二维分离模式,开展离线二维色谱分离研究,并对复杂肽段进行分离.羟丙基纤维紊(HPC)涂层的毛细管用于cIEF分离,对6种标准蛋白质的平均分离柱效约为31万.在毛细管末端引进电隔离槽,方便了第一维样品的收集.在加电6 kV下,第二维pCE...     

等电聚焦分离与18O稳定同位素标记联用的磷酸化蛋白质组学定量方法研究

磷酸化蛋白质组学定量分析,要对磷酸化修饰富集技术和定量技术进行研究。基于此,本研究采用18O稳定同位素标记技术对胰蛋白酶酶解肽段混合物进行标记,并对其标记时间和标记后胰蛋白酶的变性条件进行优化。结果表明:在pH=4～5的KH2PO4缓冲体系中,37℃,标记反应持续19～24h,除了C-端肽之外,几乎所有的肽段都可达到100%标记;采用TCEP可以有效地抑制16O-18O回标现象。建立了与18O标记技术兼容性良好的IPG-IEF技术对磷酸化肽段进行选择性富集,富集后共从HepG2细胞中鉴定到491个磷酸化位点、362个磷酸化肽段和356个磷酸化蛋白,表明IPG-IEF在大规模磷酸化肽段分离富集中是有效的;最后与高准确度高灵敏度高分辨率的LTQ-FTICR质谱仪联用,建立了基于18O-IPG-IEF-LTQ-FTICR的磷酸化蛋白质组定量技术。实验结果表明,该技术可以实现磷酸化肽段的有效定性和定量。本研究为磷酸化蛋白质组学定量研究提供了实用技术。     

毛细管等电聚焦/毛细管无胶筛分电泳二维蛋白质分离平台的构建

设计并制作了一种新型的中空纤维接口,以此为核心构建了毛细管等电聚焦(CLEF)／毛细管无胶筛分(CNGE)电泳二维蛋白质分离技术平台,实现了将二维凝胶电泳从平板转移到毛细管中。利用该二维分离平台,对血红蛋白样品进行了高效、快速分离分析,验证了该二维分离平台的可行性及分离效能。实验结果表明：二维分离系统总的峰个数和分离效能都比各自一维分离系统有较大提高。     

Serum Proteomic Analysis of Cannabis Use Disorder in Male Patients

Cannabis use has been growing recently and it is legally consumed in many countries. Cannabis has a variety of phytochemicals including cannabinoids, which might impair the peripheral systems responses affecting inflammatory and immunological pathways. However, the exact signaling pathways that induce these effects need further understanding. The objective of this study is to investigate the serum proteomic profiling in patients diagnosed with cannabis use disorder (CUD) as compared with healthy control subjects. The novelty of our study is to highlight the differentially changes proteins in the serum of CUD patients. Certain proteins can be targeted in the future to attenuate the toxicological effects of cannabis. Blood samples were collected from 20 male individuals: 10 healthy controls and 10 CUD patients. An untargeted proteomic technique employing two-dimensional difference in gel electrophoresis coupled with mass spectrometry was employed in this study to assess the differentially expressed proteins. The proteomic analysis identified a total of 121 proteins that showed significant changes in protein expression between CUD patients (experimental group) and healthy individuals (control group). For instance, the serum expression of inactive tyrosine protein kinase PEAK1 and tumor necrosis factor alpha-induced protein 3 were increased in CUD group. In contrast, the serum expression of transthyretin and serotransferrin were reduced in CUD group. Among these proteins, 55 proteins were significantly upregulated and 66 proteins significantly downregulated in CUD patients as compared with healthy control group. Ingenuity pathway analysis (IPA) found that these differentially expressed proteins are linked to p38MAPK, interleukin 12 complex, nuclear factor-κB, and other signaling pathways. Our work indicates that the differentially expressed serum proteins between CUD and control groups are correlated to liver X receptor/retinoid X receptor (RXR), farnesoid X receptor/RXR activation, and acute phase response signaling.     

液相等电聚焦结合乙酰化标记在比较蛋白质组学中的应用

建立了液相等电聚焦,一维电泳分离以及肽段乙酰化同位素标记的新技术,此技术在1∶5~5∶1范围内具有较好的动态范围(误差小于20%)。本方法不仅可以提高比较蛋白质组分离的通量,并有助于低丰度差异蛋白中的检出;并成功应用于正常肝脏与癌变肝脏组织的低丰度、偏碱性蛋白的差异比较蛋白质组分析,鉴定出16种差异蛋白,结果令人满意。     

Electrochemical Reactions During Isoelectric Focusing and Their Role in Establishment of the pH Gradient

The process occurring as the background to IEF is electrolysis of water. During IEF the yield of water ions decreases, following a non-linear relationship similar to that of the current. Different extents of acidification and basification of the electrode solutions, causing a drift of the pH gradient, were observed. We derived a relationship underlying this process which enables calculation of the electrode current. By using a novel approach we showed that the sum of the pH of distilled water electrode solutions at the end of the IEF process tends to the ionic product of water.