NanoLC-ESI MS/MS对重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白的准确鉴定

重组人Ⅱ型肿瘤坏死因子受体一抗体融合蛋白(商品名益塞普)是一种用于治疗中度及重度活动性类风湿关节炎的重组蛋白药物.该药由肿瘤坏死因子受体和人IgG1的FC片段融合而成.国内的药品报批时,没有明确规定必须为人IgG1的FC片段.但该药品出口到国际市场时,要求必须确定为人IgG1的FC片段.人IgG共有4个亚型,序列高度同源.为准确鉴定人IgG1,本实验应用超高效纳升液相色谱一高解析离子淌度质谱仪HDMS进行了分析,结果表明重组人Ⅱ型肿瘤坏死因子受体一抗体融合蛋白一级结构正确,FC片段确实为人IgG1,符合出口国际市场的标准.     

超高效纳升液相色谱-电喷雾串联质谱鉴定重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白

采用超高效纳升液相色谱-电喷雾串联质谱对重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白进行了分析.样品经胰蛋白酶酶切后,进行液相色谱-串联质谱分析和数据库检索.结果表明, 分别有5个和7个肽段匹配于人肿瘤坏死因子受体和人Ig gamma-1 chain C region.比对分析表明,重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白中检测到的人IgG的7个肽段完全与人IgG1的序列匹配,与其它3个亚型只有部分肽段匹配.说明重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白一级结构正确,Fc片段确实为人IgG1的Fc片段.     

酪氨酸激酶受体多肽片段在SPR传感芯片表面的磷酸化及其与下游蛋白的相互作用

胰岛素样生长因子-1受体(IGF-1R)信号转导通路与各种正常生理活动及多种疾病发生密切相关.将IGF-1R来源的未磷酸化多肽序列LYASVNPEY固定于表面等离子体共振(SPR)生物传感器专用的CM5芯片表面,以含有相应蛋白激酶的细胞裂解液进行处理,利用SPR生物传感器和抗酪氨酸磷酸化抗体PY20进行酪氨酸磷酸化水平检测.结果表明:PY20能灵敏地检测多肽酪氨酸磷酸化水平变化,即能够区分不同孵育时间以及不同成分的细胞裂解液所引起的多肽酪氨酸磷酸化水平差异;研究了IGF-1R来源多肽的酪氨酸磷酸化对多肽-蛋白间相互作用的影响,证实了只存在磷酸化的多肽与其下游的胰岛素受体底物(IRS-1)相互作用,测定了此磷酸化的多肽与IRS-1的亲和常数(KA)为 (2.02 ± 0.09)×108 L mol-1、结合速率常数(ka)为(2.30±0.15)×106 L mol-1s-1以及解离速率常数(kd)为(1.14±0.13)×10-2 s-1.这种新型的模拟受体的多肽酪氨酸磷酸化研究方法可用于受体磷酸化信号通路的研究以及基于这些信号通路的药物筛选研究.     

基于两相预柱的在线二维分离系统用于人肝组织中磷酸肽的规模化鉴定

将C18反相(RP)填充柱和磷酸基强阳离子交换(SCX)整体柱集成于一体的RP - SCX两相预柱,成功用于RPLC - MS/MS系统的自动进样和SCX分级,完成了磷酸肽在线多维分离平台的构建,可以减少样品的损失,极大地提高了系统的集成化水平和检测灵敏度.对人肝组织酶解液富集的磷酸肽进行规模化鉴定,在假阳率小于1％的情况下,共鉴定到3082条非重复的磷酸化肽段、3056个磷酸化位点和1332个磷酸化蛋白.对所鉴定到的磷酸化蛋白进行了功能分类和激酶分析.该实验结果对于了解蛋白质磷酸化在肝脏组织中发挥的生理学作用具有重要意义.     

基于酪氨酸磷酸酶(PTP1B)的Ⅱ型糖尿病药物设计、合成和体外活性及毒性的初步评价

酪氨酸磷酸酶PTP1B已被公认为治疗Ⅱ型糖尿病药物的理想靶标蛋白.通过重组表达制备PTP1B蛋白及其蛋白质晶体,并尝试将其与768种小分子片段进行以结构为基础的筛选,得到了数个与PTP1B结合的小分子片段及其化学结构.在此基础上利用计算机辅助药物设计方法设计与优化出一种新型PTP1B抑制剂,并经13步反应合成了该目标分子.体外药理活性和毒性的初步评价的结果表明,其对体外重组人源PTP1B的酪氨酸磷酸酶活性半数抑制浓度IC50值达到(3.4±1.2) μmol/L,优于阳性对照;在HepG2细胞胰岛素抵抗模型中,该化合物能有效改善胰岛素抵抗细胞对葡萄糖的利用能力,其改善效果与阳性药匹格列酮相当.初步的斑马鱼毒性检测表明,目标该化合物在浓度为500μmol/L时未对幼鱼产生明显毒性.新化合物的发现为新型PTP1B抑制剂的后续开发开辟了新基础.     

酵母丙氨酸转移核糖核酸双氢尿核苷(D)环区九核苷酸(14-22)的合成

用有机合成或酶促合成的AGDCGG为受体,以过量的pDAGp为供体,应用T_4RNA连接酶,成功地合成了酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))的双氢尿核苷(D)环区九核苷九磷酸AGDCGGDAGp.在脱去3’-端磷酸基团后,得九核苷八磷酸.产物经序列分析证明,与AGDCGGDAG完全符合.本文也进行了相应于D环区九核苷酸的组成片段AG,AGDC,GG,GGp和DAG的5’-磷酸化和它们之间连接的条件探索,发现了3’-DMP和DAG的5’-磷酸化需在5～10℃下进行,可获近定量的产率. AGDCGGDAGp已用于tRNA_y~(Ala)的全分子合成.     

壳寡糖和磷酸化壳寡糖对Cu(Ⅱ)的络合和缓释性能

以壳寡糖(COS)和磷酸化壳寡糖(PCOS)为原料与铜离子反应,制备了壳寡糖铜(Ⅱ)络合物(COS-Cu)和磷酸化壳寡糖铜(Ⅱ)络合物(PCOS-Cu),讨论了pH、时间、温度和PCOS取代度对络合物吸附量的影响.释放性能表明COS-Cu(Ⅱ)和PCOS-Cu(Ⅱ)均具有缓释性能,且PCOS-Cu(Ⅱ)具有更加均匀的释放速率.     

大鼠肝脏金属硫蛋白的分离纯化和金属硫蛋白亚型-Ⅱα结构域片段的制备及其核磁共振鉴定

Wistar雄性大鼠经腹腔注射CdCl_2诱导其肝脏合成金属硫蛋白。取鼠肝,先经匀浆、热变性、乙醇-氯仿萃取、丙酮沉淀等步骤,后由Sephadex G-75、DEAE-52柱层析,得金属硫蛋白的两亚型Ⅰ和Ⅱ。经原子吸收光谱测定每蛋白分子含有5个镉和2个锌。用枯草杆菌酶割法制备并分离了金属硫蛋白亚型Ⅱ的α结构域片段。还用NMR研究了金属硫蛋白的两个亚型及所得的α结构域片段。     

肝素与HIV-1膜表面蛋白gp120相互作用的预测和模拟

用分子模拟软件研究肝素与HIV-1膜表面蛋白gp120的相互作用.将肝素中的单糖、二糖和三糖片段作为探针对gp120蛋白进行搜索,统计分析确定肝素结合区域.用肝素六糖片段和结合区域进行反应分子对接,获得两种结合模式.最终建立的模型能够很好地解释肝素体外抑制HIV-1的现象,同时对其机理进行了推测.     

基于纳米结构材料的磷酸化蛋白/多肽富集和分析

蛋白质磷酸化是最为广泛的翻译后修饰之一。在生物体液或组织中,许多低丰度的磷酸化蛋白和磷酸化肽是具有高度临床灵敏性和特异性的生物标记物,这些生物分子对许多疾病的检测和病理的阐释可能提供重要的信息。因为蛋白质磷酸化动态可逆且磷酸化蛋白丰度很低,所以很难直接从复杂的生物样品中直接检测到磷酸化蛋白和磷酸化肽。纳米结构材料因其大比表面积、丰富的活性亲合位点和特殊结构,在磷酸化肽和磷酸化蛋白的分离和富集方面已经引起了特别的关注,并成为目前磷酸化蛋白质组学富集和鉴定方面的研究热点。许多介孔、磁性、杂化或化学修饰的亲合材料被研发并用于磷酸化蛋白/多肽的富集与分离;此外,一些多功能纳米结构材料也被研发并用于蛋白质组学中磷酸化蛋白/多肽的快速高效的富集提纯。在这篇综述中,我们专注于纳米结构材料在磷酸化蛋白/多肽富集和提纯方面的最新进展。     

A 2D NMR method to study peptide phosphorylation

We demonstrate a 2D NMR method which distinguishes between phosphorylated and non-phosphorylated amino acids. The method is capable of monitoring the amino acid and site-specific enzymatic phosphorylation and dephosphorylation of peptides. The method was developed using O-phosphorylated amino acids and its potential is shown with a peptide fragment of the myelin basic protein (MBP).     

Oxidative modifications of the Photosystem II D1 protein by reactive oxygen species: from isolated protein to cyanobacterial cells

Action of reactive oxygen species (ROS) on the isolated D1 protein, a key component of Photosystem II (PSII) complex, was studied and compared with the effect of high irradiance on this protein in mildly solubilized photosynthetic membranes and cells of the cyanobacterium Synechocystis. Whereas singlet oxygen caused mainly protein modification reflected by shift of its electrophoretic mobility, action of hydrogen peroxide and superoxide resulted in generation of specific fragments. Hydroxyl radicals as the most ROS induced fast disappearance of the protein. The results substantiate the ability of ROS to cause direct scission of the D1 peptide bonds. Similar D1 modification, fragmentation and additionally cross-linking with other PSII subunits were observed during illumination or hydrogen peroxide treatment of mildly solubilized thylakoids. Peroxide-induced fragmentation did not occur in thylakoids of the strain lacking a ligand to the nonheme iron, confirming the role of this prosthetic group in the D1-specific cleavage. The D1 modification, fragmentation and cross-linking were suppressed by ROS scavengers, supporting the direct role of ROS in these phenomena. Identical symptoms of the ROS-induced D1 damage were detected in illuminated cells of Synechocystis mutants with a higher probability of ROS formation, documenting the relevance of the in vitro results for the situation in vivo.     

Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite

The formation of biogenic materials requires the interaction of organic molecules with the mineral phase. In forming enamel, the amelogenin proteins contribute to the mineralization of hydroxyapatite (HAp). Leucine-rich amelogenin protein (LRAP) is a naturally occurring splice variant of amelogenin that comprises amelogenin's predicted HAp binding domains. We determined the partial structure of phosphorylated and non-phosphorylated LRAP variants bound to HAp using combined solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. New ssNMR measurements in the N-terminus indicate a largely extended structure for both variants, though some measurements are consistent with a partially helical N-terminal segment. The N-terminus of the phosphorylated variant is found to be consistently closer to the HAp surface than the non-phosphorylated variant. Structure prediction was biased using 21 ssNMR measurements in the N- and C-terminus at five HAp crystal faces. The predicted fold of LRAP is similar at all HAp faces studied, regardless of phosphorylation. Largely consistent with experimental observations, LRAP's predicted structure is relatively extended with a helix-turn-helix motif in the N-terminal domain and some helix in the C-terminal domain, and the N-terminal domain of the phosphorylated variant binds HAp more closely than the N-terminal domain of the non-phosphorylated variant. Predictions for both variants show some potential binding specificity for the {010} HAp crystal face, providing further support that amelogenins block crystal growth on the a and b faces to allow elongated crystals in the c-axis.     

Phosphopeptide analysis by positive and negative ion matrix-assisted laser desorption/ionization mass spectrometry.

This article describes a simple procedure for the detection of phosphorylated peptides by comparable positive and negative ion mode matrix-assisted laser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresponding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their non-phosphorylated analogs. The effect was successfully used to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta-casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positive and negative) ion modes. The comparison of positive and negative ion spectra of a given protein digest (relative intensity([M - H]-)/relative intensity([M + H]+)) can be used to identify any phosphopeptides present which may then be separated and analyzed further.     

Label-free electrochemical detection of the phosphorylated and non-phosphorylated forms of peptides based on tyrosine oxidation

Identification of protein phosphorylation is an important goal in proteomics, because of the central role played by phosphorylation in the regulation of cellular activities. An exciting consequence of tyrosine (Tyr) oxidation is that it allows a clear distinction between the phosphorylated and non-phosphorylated forms of peptides using electrochemical analysis. In this report, we monitored the effect of phosphorylation on the electro-oxidation of Tyr in connection with differential pulse voltammetry (DPV) using a screen-printed carbon electrode (SPCE). First, we monitored the electrochemical current responses of Tyr and o-phospho-l-Tyrosine (l-3-(4-hydroxyphenyl)alanine 4′-phosphate, Tyr-P). The detection limit for Tyr was determined as 10 nM on the SPCE surface (S/N = 3). We observed that the phosphorylation caused a significant suppression on the electro-oxidation of Tyr. We also monitored the electrochemical responses of Src peptide 521–533 (H–Thr-Ser-Thr-Glu-Pro-Gln-Tyr-Gln-Pro-Gly-Glu-Asn-Leu–OH), both in the non-phosphorylated and phosphorylated forms. The detection limit for Src peptide was determined as 100 nM (S/N = 3). By monitoring the current signals obtained from the Tyr kinase substrate peptides, we suggest that label-free electrochemical in vitro detection of Tyr phosphorylation can be performed in a rapid and cost-effective format.     

Label-free electrochemical differentiation of phosphorylated and non-phosphorylated peptide by electro-catalyzed tyrosine oxidation

Protein phosphorylation plays an important role in many significant cellular processes, and has thus gained tremendous interest in the field of proteomics. The electro-active tyrosine residue, as an important receptor of phosphorylation in proteins, exhibits electro-inactivity after being phosphorylated on the hydroxy group of its aromatic ring. In this study, the electrochemical oxidation of tyrosine on indium tin oxide (ITO) electrodes was catalyzed with an electron mediator Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine) and was employed as a signal reporter to differentially detect non-phosphorylated and phosphorylated peptides. A short, tyrosine-containing peptide glu-glu-glu-glu-glu-tyr (EY-6) was immobilized on an ITO surface using the layer-by-layer self-assembly method, and was detected by cyclic voltammetry in an Os(bpy)(3)(2+) solution. The limit of detection was about 0.23 microg mL(-1) EY-6 in solution. The phosphorylated peptide glu-glu-glu-glu-glu-tyr-OP (EY-6P) did not produce an appreciable oxidation current on the electrode. Surface plasmon resonance measurements revealed that the amount of EY-6 and EY-6P adsorbed on the sensor chip surface was 269 and 378 pg mm(-2), respectively. The poly(glu, tyr) (4 : 1) peptide, a protein tyrosine kinase substrate, was also detected by the same approach, with a detection limit of 0.65 microg mL(-1). This new approach offers the possibility of label-free and on-chip detection of protein tyrosine kinase activity.     

An integrated procedure of selective injection, sample stacking and fractionation of phosphopeptides for MALDI MS analysis

Protein phosphorylation is one of the most important post-translational modifications (PTM), however, the detection of phosphorylation in proteins using mass spectrometry (MS) remains challenging. This is because many phosphorylated proteins are only present in low abundance, and the ionization of the phosphorylated components in MS is very inefficient compared to the non-phosphorylated counterparts. Recently, we have reported a selective injection technique that can separate phosphopeptides from non-phosphorylated peptides due to the differences in their isoelectric points (pI) [1]. Phosphorylated peptides from α-casein were clearly observed at low femtomole level using MALDI MS. In this work, further developments on selective injection of phosphopeptides are presented to enhance its capability in handling higher sample complexity. The approach is to integrate selective injection with a sample stacking technique used in capillary electrophoresis to enrich the sample concentration, followed by electrophoresis to fractionate the components in preparation for MALDI MS analysis. The effectiveness of the selective injection and stacking was evaluated quantitatively using a synthetic phosphopeptide as sample, with an enrichment factor of up to 600 being recorded. Next, a tryptic digest of α-casein was used to evaluate the separation and fractionation of peptides for MALDI MS analysis. The elution order of phosphopeptides essentially followed the order of decreasing number of phosphates on the peptides. Finally, to illustrate the applicability, the integrated procedure was applied to evaluate the phosphorylation of a highly phosphorylated protein, osteopontin. Up to 41 phosphopeptides were observed, which allowed us to examine the phosphorylation of all 29 possible sites previously reported [2]. A high level of heterogeneity in the phosphorylation of OPN was evident by the multiple-forms of variable phosphorylation detected for a large number of peptides.     

基于生物质谱技术的磷酰化修饰策略在多肽测序中的应用

该文建立了一种利用磷酰化修饰结合电喷雾质谱(ESI-Q-TOF)测定多肽氨基酸序列的有效方法。利用Atherton-Todd反应,以二丙基亚磷酰酯(DPP)为磷酰化试剂,应用生物质谱技术,对磷酰化修饰后的5种模型肽的磷酰化反应情况进行了系统研究,考察了磷酰化肽的二级质谱特征,并与未经磷酰化反应的肽的二级质谱特征对比。结果表明,经过磷酰化修饰后,肽的二级质谱中的a1离子信号强度明显增加,可以准确鉴定其N端氨基酸;b系列离子信息完整,信号强度增强,使得多肽C ID测序的谱图简单、清晰,有利于肽的氨基酸序列的测定;赖氨酸(K,128.10 u)和谷氨酰胺(Q,128.13 u)两种氨基酸质荷比相近,由于二者磷酰化修饰后的差异性,使其得到准确区分。经过5种已知氨基酸序列的模型肽的磷酰化后结合质谱技术进行氨基酸序列测定验证,结果表明该方法简单、快速、准确,提高了利用质谱技术进行多肽测序的准确度和灵敏度,可为蛋白质组学研究提供有效的技术手段。     

Phosphorylation of phospholipase D1 and the modulation of its interaction with RhoA by cAMP-dependent protein kinase

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.     

Analysis of phosphorylation sites on focal adhesion kinase using nanospray liquid chromatography/multiple reaction monitoring mass spectrometry

An approach based on nanospray liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) was developed in order to analyze twenty-nine phosphorylated and non-phosphorylated tryptic peptides from focal adhesion kinase (FAK). All peptides monitored were resolved and showed excellent peak shape with the exception of one doubly phosphorylated peptide. Optimization of the LC method enabled the identification and subsequent monitoring of six important tyrosine phosphorylation sites on FAK, including phosphorylated Y397 (pY397), pY407, pY576, pY577, pY861, and pY925. This technique was able to identify sites of phosphorylation on FAK as well as qualitatively differentiate between autocatalytic and Src-induced phosphorylation events. FAK was shown to have autocatalytic function, which resulted in efficient phosphorylation of Y397. FAK was also capable of autophosphorylation on residues Y407 and Y576, though apparently less effectively than autophosphorylation at Y397. Src was found to phosphorylate FAK at Y407, Y576, Y577, and Y861. The presence of Src increased the abundance of pY576 at low temperature indicating Src had particularly high kinase activity toward this residue. Furthermore, Src phosphorylated FAK at Y577 to produce FAK bis-phosphorylated at Y576 and Y577. In addition, six novel sites of phosphorylation (Y148, Y347, Y441, T503, S850, and Y1007) were identified on FAK. Interestingly, Src phosphorylated FAK to form a peptide uniquely phosphorylated on Y407, together with substantial amounts of the bis-phosphorylated pY397pY407 peptide. These findings will impact significantly on future studies of FAK activity since pY397 is often used as a measure of FAK activity and Src association.