反相高效液相色谱/电喷雾质谱法分析化学合成七肽粗产物

利用RP—HPLC／ESI—MS直接分析用芴甲氧羰基(Fmoc)固相多肽合成方法在PHB树脂上偶联合成的一个七肽(H2N—Tyr—Val—Asn-Thr-Asn—Met—Gly—COOH,Mr797．3)粗产物。RP—HPLC显示合成粗产物含有1个主成分,4个次要成分和多个微量成分;与之联用的电喷雾质谱则同步准确地测定出各成分的分子量(m／z)并自动对各主要成分的化学结构进行了串联质谱分析。结果证明,粗产物中的主成分即为目标七肽,另外几个主要副产物为七肽的氧化产物或残缺肽。     

化学合成虎纹捕鸟蛛毒素-Ⅰ的色谱行为及分离纯化

 利用反相高效液相色谱 (RP HPLC)和离子交换色谱 (IEC)比较研究了固相化学合成的多肽类神经毒素虎纹捕鸟蛛毒素 Ⅰ (HWTX Ⅰ )与其对应的天然产物的色谱行为差异及分离纯化方法。发现在完全相同的实验条件下 ,复性处理后的合成HWTX Ⅰ的谱峰有别于还原变性后又复性的天然HWTX Ⅰ的谱峰 ,主要表现为扩散度较大 ,对称性较差 ,说明合成产物存在更大分子构象的不均一性。推测除了在合成HWTX Ⅰ复性过程中有少数分子发生二硫键错配外 ,还有部分分子在固相合成中发生了消旋作用 ,需要交替采取多种色谱法才能完全分离纯化复性后的合成多肽产物。     

液相色谱-质谱联用技术分析固相合成胸腺五肽及其副产物

利用反相高效液相色谱(RP-HPLC)与电喷雾质谱(ESI-MS/MS)联用技术,分析用芴甲氧羰基(Fmoc)固相合成方法在WANG树脂上手工合成的胸腺五肽(H2N-Arg-Lys-Asp-Val-Tyr-COOH)粗产物,RP-HPLC结果显示:合成粗产物含有一个主要成分,三个次要成分和多个微量成分;与之联用的电喷雾质谱同步得出相应的某些信息,可对各成分的结构进行分析。结果证明,粗产物中的主成分即为目标五肽,另外几个主要副产物为五肽合成过程中去保护未完全的副产物。     

液质联用对半边旗二萜类活性成分分离富集方法的研究

运用高效液相色谱-质谱联用技术研究了以大孔吸附树脂富集分离,并以AgNO3-硅胶柱分离纯化半边旗二萜类活性成分的方法．采用[M—H]^-为选择监测离子,以色谱保留时间和选择监测离子流峰面积跟踪监测目标成分,探讨最佳分离条件;以萃取产物的总离子流图(TIC)和质谱图评价提取产物的纯度。研究表明,大孔吸附树脂对5F和4F具有一定的分离富集作用;AgNO3-硅胶柱可以分离纯化5F。本实验对复杂基体中化学结构上仅相差1个双键、相对分子质量相差2的5F和4F(无紫外吸收)进行了分析鉴定,获得满意结果。     

反相高效液相色谱-电喷雾质谱法分析食源血管紧张素转换酶抑制肽

利用反相高效液相色谱/电喷雾离子阱质谱法,直接分析从牦牛乳酪蛋白中酶水解得到的血管紧张素转换酶抑制肽粗产物。RP-HPLC显示具有活性的多肽粗产物含有3个主要成分,质谱同步测定各组分的分子量(m/z)分别为815.2,1680.1,962.2,然后选择[M H] 离子通过串联质谱(MS/MS)得到碎片离子,利用b离子和y离子互补的方法鉴定了多肽序列。三条肽分别为Leu-Pro-Tyr-Tyr,Pro-Leu-Pro-Leu-Leu-Gln,Phe-Leu-Pro-Pro-Tyr-Tyr。结果显示,所获得的多肽序列与牛乳酪蛋白一级结构中相应肽段的序列一致。     

合成多肽的电喷雾质谱研究

利用电喷雾多极串联质谱对3种合成多肽进行了系统的鉴定和分析研究。首先通过全扫描模式测定了其分子量，然后选择[M H]^ 或[M 2H]^2 离子通过串联质谱(MS／MS)得到碎片离子，采用y离子和b离子互补的方法测定了多肽序列。利用文献数据对这种方法进行了验证，实验结果表明，该方法简便、快速、实用。     

利用高效液相色谱鉴定化学合成的 δ-睡眠诱导肽及其类似物

用高效液相色谱对化学方法合成的δ-睡眠诱导肽(DSIP)及其类似物(Tyr~1-DSIP,Phe~5-DSIP以及Tyr~1Phe~5-DSIP)的主要成份进行了分离,应用在线的紫外光谱和萤光光谱检测,并将分离出的色谱峰收集后进行氨基酸分析。     

维生素E辛烯基琥珀酸酯的定性分析

通过比较不同高效液相色谱(HPLC)方法对维生素E与辛烯基琥珀酸酐反应产物分离效果的影响,最终建立了以Dionex Kromasil C18柱为分析柱、甲醇和0.033 mol/L H3PO4(100:1,体积比)为流动相的色谱分离条件;借助高效液相色谱-大气压化学电离源质谱联用(HPLC-APCI-MS)法检测分离得到的色谱峰,通过比较它们的紫外光谱和质谱特征,确定其中两种物质为维生素E辛烯基琥珀酸酯的异构体。     

硒芳香杂环化合物的GC/MS分析

本文对合成的7种含硒芳香杂环化合物进行了GC／MS分析研究。结果表明：BS、MB、BBS和DBBS等4个化合物在色谱柱内的保留时间与它们的相对分子质量呈线性关系。所有化合物均可获得特征质谱,表现出含单个硒原子的分子离子或碎片离子特征峰簇,硒的两种主要同位素在峰簇中表现为主要峰M与（M-2）的相对丰度比约为2：1,可为鉴定含硒分子离子或碎片离子提供重要信息。新化合物1,2,5-硒二唑并[3,4-d]嘧啶-5,7-（4H,6H）二酮（SPDO）在色谱柱内出现11．83min和7．96min两个具有相同的质谱的色谱峰,被认为是互变异构体的峰。     

改进的~(18)O同位素标记法标记蛋白质多肽

针对18O同位素标记反应两个重要影响因素——肽段分散度和胰酶灭活方法,进行了标记条件的改进和灭活方法的优化。在H218O中加入RapigestTM SF助溶剂并微波辅助加热,使α-酪蛋白胰酶酶切肽段的标记效率得到明显改进(18O/16O峰面积比值>99%)。标记后,对胰酶进行还原烷基化化学修饰彻底灭活,使标记后的肽段稳定性显著提高,放置6d不发生回交反应。对标准蛋白质甲状腺球蛋白酶切肽段混合物标记后的质谱实验结果表明:优化的标记方法能快速稳定地标记蛋白质酶切多肽。     

多肽新药研发策略研究进展

多肽药物具有生物活性高、药用剂量小、产业化开发优势明显等诸多优点,已成为全球关注的创新药物研发热点之一.但是其代谢不稳定、半衰期短及较难通透组织屏障等缺点严重阻碍了多肽新药在临床治疗中的广泛应用.为了解决这些限制多肽药物的瓶颈,本课题组发展了一系列的改造策略.通过这些策略的应用,以期加快多肽药物临床应用的步伐.本文主要结合本课题组的工作对多肽新药创制过程中所遇到的关键问题及解决思路进行综述.     

Response of peptide intensity to concentration in ESI–MS-based proteome

High-throughput quantification with label-free methods has received considerable attention in electrospray ionization(ESI)-mass spectrometry(MS),but the manner by which MS signals respond to peptide concentration remains unclear in proteomics.We developed a new mathematical formula to describe the intrinsic log-log relationship between the MS intensity response and peptide concentration in an analytical ESI process.Experimental results showed that the calibration curve is fairly fit to the log-log formula with a linear dynamic range of approximate four to five orders of magnitude.However,we found that the ionization of analytical peptides can be severely suppressed by coexisting matrix peptides,such that the calibration curve can be poorly leveled off on both ends.Our study suggests that the interferences from coexisting matrix peptides should be reduced in the ESI process to use the log-log calibration curve successfully for the high-throughput quantification.     

Structures of the Peptide Leu-Pro-Tyr-Pro and Its Derivatives and the Nicotinamide Part of NADPH by a Semi-Empirical PM3 Method

The capability of peptides Leu-Pro-Tyr-Pro (LPYP), Leu-Pro-Tyr-Pro-Arg (LPYPR), and Ser-Pro-Tyr-Pro-Arg (SPYPR) to occupy the part of the binding site ascribed to NADPH in the active center of 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase was analyzed using results from a semi-empirical PM3 method. The similarity of the peptide structures to NADPH was determined by comparing the relative contribution from projections of selected bond lengths in the peptides in two mutually perpendicular planes to the corresponding bond lengths in the nicotinamide part of the substrate. The correlation coefficient between the calculated average values of the relative contributions of the bonds and the inhibitory activity of these peptides is rather high (R = 0.926). This indicates that these peptides can occupy the part of the binding site for NADPH in the active center of the enzyme.__________Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 56–59, January–February, 2005.     

多肽片段连接方法及肽硫酯和肽醛的合成*

本文综述了多肽和蛋白质合成中的片段连接方法,这是近年来多肽和蛋白质合成领域中方法学上的重要进展.该方法使用非保护的多肽片段,无需酶或化学活化试剂,在缓冲溶液中能够高产率地获得多肽和蛋白质.还介绍了与多肽片段连接有关的肽硫酯和肽醛的合成方法.     

HF‐Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post‐translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.     

The Synthesis of a Novel Phosphorus Containing Antigen

The hydrolysis of peptide is one of the most important chemical processes in life chemistry. It is of great significance to study catalytic antibody, which is capable of catalyzing the hydrolysis on a specific peptide bond. In recent years, we have successfully synthesized some tetrahedral geometry analogues mimicking that of the hippuryl phenylalanine 1 hydrolyzing transition state catalyzed by carboxypeptidase A(CPA)1,2, which is specific for cleavage of the C-terminal amino acid from an o…     

钳蝎毒中抗癫痫肽、镇痛肽和抗肿瘤肽的快速同时分离和鉴定

采用Shim-Pack WCX-1型阳离子交换高压色谱柱对中国东亚钳蝎全蝎毒进行了分离，在鉴定了其中的抗癫痫肽、镇痛肽和抗肿瘤肽活性峰的基础上，应用Shim-Pack DIOL-300型凝胶排阻高压色谱柱对它们进行了进一步分离和鉴定，可以得到较纯的3种多肽。在高压色谱所提供的全蝎毒分离信息的基础上，应用与Shim-Pack WCX-1色谱柱具有相同交换基团的、具有较大吸附容量的CM Sepharose CL-6B软胶介质在低压色谱上对全蝎毒进行了分离，并分别对其中的抗癫痫肽、镇痛肽和抗肿瘤肽进行了鉴定。     

PepPro: A Nonredundant Structure Data Set for Benchmarking Peptide–Protein Computational Docking

We present a nonredundant benchmark, coined PepPro, for testing peptide–protein docking algorithms. Currently, PepPro contains 89 nonredundant experimentally determined peptide–protein complex structures, with peptide sequence lengths ranging from 5 to 30 amino acids. The benchmark covers peptides with distinct secondary structures, including helix, partial helix, a mixture of helix and β-sheet, β-sheet formed through binding, β-sheet formed through self-folding, and coil. In addition, unbound proteins' structures are provided for 58 complexes and can be used for testing the ability of a docking algorithm handling the conformational changes of proteins during the binding process. PepPro should benefit the docking community for the development and improvement of peptide docking algorithms. The benchmark is available at http://zoulab.dalton.missouri.edu/PepPro_benchmark . © 2019 Wiley Periodicals, Inc.     

Diaminodiacid Bridges to Improve Folding and Tune the Bioactivity of Disulfide‐Rich Peptides

Disulfide‐rich peptides containing three or more disulfide bonds are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low‐yielding folding process. We found that a single cystine‐to‐diaminodiacid replacement could significantly increase the folding efficiency of disulfide‐rich peptides and thus improve their production yields. The practicality of this strategy was demonstrated by the synthesis and folding of derivatives of the μ‐conotoxin SIIIA, the preclinical hormone hepcidin, and the trypsin inhibitor EETI‐II. NMR and X‐ray crystallography studies confirmed that these derivatives of disulfide‐rich peptide retained the correct three‐dimensional conformations. Moreover, the cystine‐to‐diaminodiacid replacement enabled structural tuning, thereby leading to an EETI‐II derivative with higher bioactivity than the native peptide.     

O8肽的HPLC分析与制备研究

系统建立了化学合成小分子肽(08肽)的反相HPLC分离分析方法．考察了色谱填料种类、流动相组成及梯度条件对样品分离的影响．通过从分析型到半制备型分离线性放大过程中分离条件与分离效果的关系的探讨．建立了等度洗脱条件下较大规模地分离制备该小肽的方法试验结果表明：硅胶基质的色谱柱比聚合物基质的色谱柱更适合于分离该小肽,而粒径(5μm．10μm)对分离的影响不大;较缓的梯度条件可以明显改善分离,但所需分离时间延长一在制备分离中应综合考虑分离效果与运行时间的关系。