蛋白质及多肽的液相色谱-电喷雾离子化质谱研究进展

蛋白质及多肽的液相色谱电喷雾离子化质谱研究进展徐友宣彭师奇（北京医科大学生源药物化学中德联合实验室，北京１０００８３）多肽及蛋白质药物对临床的重要性，已引起各国药物化学界的广泛重视。在多肽或蛋白质药物的结构研究中，ＭＳ研究有重要价值。例如ＦＡＢＭ...     

猪脑钠素及其类似物的合成

以固相多肽合成方法合成了猪的二十六肽脑钠素(BNP)和它的一个类似物(Mpr⁴,D-Ala^6,13)-BNP(4-24)-NH₂,保护肽用HF裂解除去保护基、在碱性条件下空气氧化形成二硫桥后,粗产物经凝胶过滤和高效液相色谱分离纯化,均有与天然脑钠素相同的活性。合成肽相对于树脂初始取代量的产率分别为9.56%和11.03%。     

液相色谱-质谱/串联质谱联用技术及其在药物分析中的应用

液相色谱-质谱联用技术结合了色谱、质谱两者的优点，将色谱的高分离性能和质谱的高鉴别特点相结合，组成了较完美的现代分析技术。本文介绍近年来液相色谱-质谱接口技术的研究进展及液相色谱-质谱／串联质谱联用技术等在药物分析中的应用，包括药物研究的分离与鉴定，药物动力学研究的体内浓度分布、代谢物分析等，展示了它的发展前景。随着液相色谱-质谱接口技术的不断完善，液质联用技术将在药物分析中占据越来越重要的地位。     

超高效液相色谱（UPLC）在药物分析领域中的应用

超高效液相色谱（Ultra Performance Liquid Chromatography，UPLC）是近年来发展迅速的基于小颗粒填料的液相色谱技术，既能缩短分析时间，又可减少溶剂消耗。与普通高效液相相比，其柱效及分离能力随着使用1．7μm颗粒度的色谱柱填料得到很大提高。本文对超高效液相色谱近年在药物分析中的应用进行了综述。     

高效液相色谱法的进展及其在生化医药方面的应用

高效液相色谱由于对挥发性小或无挥发性、热稳定性差、极性强，特别是那些具有某种生物活性的物质提供了非常合适的分离分析环境，因而广泛应用于生物化学、药物、临床等。目前它已成为人们在分子水平上研究生命科学的有力工具；同样，生命科学研究的进一步深入，也推动了高效液相色谱技术的飞速发展。介绍了高效液相色谱法的特点及其在生化医药方面的应用，并对国内外高效液相色谱技术的现状、发展与动态作了分析。     

超高效液相色谱在药物分析中的应用

超高效液相色谱（UPLC）是近年来发展的以超高速度、超高分离度、超高灵敏度为特点的新型液相色谱技术。其应用于复杂体系快速分析所表现出的优势,已引起了药物分析工作者的重视。本文对近年来超高效液相色谱在药物分析中的应用进行了综述。     

制备型高效液相色谱在中药野菊花化学成分分离中的应用

目的对野菊花的黄酮类化学成分进行分离研究,并探索制备液相色谱在分离制备植物药化学对照品的研究中的应用前景。方法用硅胶及聚酰胺柱色谱进行分离,制备型高效液相色谱进一步分离纯化,通过现代光谱分析技术,确定了化合物结构。结果分离纯化了6个化合物,纯度均为99%以上。鉴定了其中三个化合物,分别为蒙花苷(Ⅰ),绿原酸(Ⅱ),咖啡酸(Ⅲ),并确定为野菊花药材含量测定的指标性成分。结论建立了蒙花苷,绿原酸,咖啡酸化学对照品的制备工艺方法,使用制备型高效液相色谱进行分离纯化,可制备量大,简便易行,在天然植物药化学对照品的制备纯化中已成为重要的手段。     

固相萃取技术在核苷类逆转录酶抑制剂定量分析研究中的应用概况

固相萃取(Solid-phase extraction,SPE)是近20年来迅速发展起来的一种生物样本预处理技术,是以液相色谱分离机制为基础建立的分离纯化方法。由于生物样品干扰成分较多且其中药物浓度多数较低,应用高效液相色谱(HPLC)和液相色谱/质谱联用(LC/MS)法往往无法实现样品的直接测定分析。SPE与HPLC或LC/MS法联用实现了样品预处理和分离分析的优化组合,在生物样本分析方面具有广泛的适应性和优越性。核苷类逆转录酶抑制剂(NRTIs)属核苷类似物,极性较强,故此联用技术在其生物样本分析测定方面应用普遍。为此,本文介绍了近年SPE与HPLC或…     

胸腺肽β4分离与纯化工艺的研究

目的:以小牛胸腺为原料,研究其中的活性多肽胸腺肽β4的分离和纯化工艺.方法:采用硫酸铵沉淀法制得胸腺素5粗提品,然后利用阴阳离子交换色谱纯化得到胸腺肽β4产品,并采用SDS-PAGE和HPLC法进行分析鉴定.结果:分离纯化得到了纯度为90.1%的产品.结论:本实验采用低压离子交换色谱分离得到了纯度为90.1%的产品,可用于进一步的工业化研究.     

液相色谱-质谱联用技术在药物分析中的应用

液相色谱-质谱（LC—MS）联用技术体现了色谱和质谱优势的互补,将色谱的高分离性能和质谱的鉴别力强的特点相结合,组成了较完美的现代分析技术。本文介绍近年来液-质联用技术在药物分析研究领域的应用,随着液相色谱一质谱质联用技术的不断完善、发展,必将在药物分析中占据越来越重要的地位。     

Hydrophilic interaction/cation-exchange chromatography for the purification of synthetic peptides from closely related impurities: serine side-chain acetylated peptides

Abstract: Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEC) is a novel HPLC technique which has excellent potential for peptide separations. Separations by HILIC/CEC are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlayed on ionic interactions with the cation-exchange matrix. Complex peptide mixtures produced by solid-phase synthesis are a frequently encountered and challenging purification problem. In the present study a two-step protocol, consisting of HILIC/CEC followed by RPC, was required for the successful purification of a 21-residue synthetic amphipathic α-helical peptide from serine side-chain acetylated impurities, with HILIC/CEC proving to be highly sensitive to subtle differences in hydrophilicities between the acetylated peptides and the desired product. Investigation of the three potential sites of serine acetylation through solid-phase synthesis of acetylated analogues of the desired peptide (peptides of the same sequence and secondary structure, but acetylated at different positions on the hydrophilic face of the α-helix) demonstrated that acetylation was occurring at different sites on the peptide. HILIC/CEC was able to take advantage of very subtle changes in environment around the acetylation sites and thus effect a separation of these analogues not achievable by RPC or CEC alone.     

Solid‐phase precipitation and extraction,a new separation process applied to the isolation of synthetic peptides

Abstract: A new method for separation and purification is described. The process, referred to as solid‐phase precipitation and extraction (SPPE), was developed and applied to postcleavage isolation of synthetic peptides. The technique uses normal approaches of chromatography and solid‐phase extraction sorbents with a precipitation or drying procedure so that the sorbent becomes a support matrix for thin‐film deposition of the compounds of interest. This procedure causes precipitated compounds of interest to be trapped on the large surface area or in the pores of the matrix so that by‐products and impurities can be removed by strong wash solvents. In application to solid‐phase peptide synthesis chemistry, by‐products from the cleavage and deprotection are selectively extracted from the crude sample mixture under mild conditions. In comparison to the ether precipitation method used in peptide chemistry, the SPPE process provides isolated products that are 14–17% (w/w) higher purity.     

Reversed phase thin-layer chromatography for the separation of β-endorphin, β-lipotropin and enkephalins

A novel, simple technique using reversed phase thin-layer chromatography has been developed to separate β-endorphin, β-lipotropin and Met-and Leuenkephalins. This method is useful for the rapid determination of the purity of these opioid peptides. The effectiveness of several solvent systems has been assessed. The resolution between β-endorphin and β-lipotropin achieved by this method exceeds that reported using high performance liquid chromatography or a variety of column techniques. This system also eliminates the problem of unrecognized loss of peptide due to binding on HPLC or other types of columns since the entire stationary phase is visualized using fluorescamine detection. Efficient, inexpensive and reliable simultaneous separation with visualization of several opioid peptides, their peptide precursors and their degradation products is now possible using the new technique. Such separations are required for many purposes including preparation of samples for radioimmunoassay of opioid peptides.     

Convergent solid-phase peptide synthesis 12. * Chromatographic techniques for the purification of protected peptide segments

The purification of a range of protected peptide segments has been carried out using modified reversed-phase chromatographic techniques in which DMF was added to the water and acetonitrile mixtures used as eluents. The purity of the recovered peptides was excellent and recoveries were high in all cases, even for longer hydrophobic segments. In several cases purifications were carried out on the hundreds of milligrams scale. For protected peptide segments containing Met, protection as the sulfoxide avoids its unwanted alkylation and oxidation, and the increased overall polarity can be useful in the purification of protected peptides incorporating this residue. © Munksgaard 1995.     

Design of ligands for the purification of anti-MUC1 antibodies by peptide epitope affinity chromatography

Abstract: The fine specificity of epitope recognition of the anti-MUCl mucin monoclonal antibody, C595 has been studied using solid-phase replacement net (RNET) analysis. Two peptides (RAAP and RPPP) showed increased reactivity with C595 antibody compared with the native epitope (RPAP). These were synthesized as integral motifs within MUCl immunodominant peptides and analyzed by fluorescence quenching (FQ) and circular dichroism (CD). They were also tested as ligands for the purification of C595 antibody using epitope affinity chromatography. Affinity matrices were compared with respect to capacity, affinity, and quality of the purified product. In FQ tests the native epitope peptide (APDTRPAPG) and the alanine substituted peptide had similar association constants when reacting with C595 antibody, whereas the proline substituted peptide (APDTRPPPG) had a higher association constant. This order of affinity for C595 was confirmed in chromatography experiments in which antibody was eluted from the former two peptide matrices at approximately the same point on the NaSCN elution gradient, whereas antibody was desorbed from APDTRPPPG at a higher NaSCN concentration. Circular dichroism analysis showed that the thermodynamically preferred conformation of these peptides in aqueous solution is the P-II extended helix, the conformation preferred for an extended bound form of the peptide held by interactions with the peptide amides. The stronger binding peptide (APDTRPPPG) has the higher population of the P-II helix in solution. In conclusion, RNET analysis is useful in the rational design of peptide ligands so that the performance of affinity matrices may be regulated.     

色谱及其联用技术在海洋药用生物核苷类化合物筛选排重方面的应用

为了解决天然产物化学研究中核苷类化合物重复提取、分离、纯化和表征等问题,避免人力物力的大量消耗并提高新化合物的发现效率,因此对已知核苷类化合物的快速鉴别、排重显得尤为重要。本文以笔者所在课题组取得的研究成果为例,从高效液相色谱、高效液相色谱-质谱联用、毛细管电泳和毛细管电泳-质谱联用等4种色谱及其联用技术,对海洋生物中核苷类化合物快速鉴别、排重方法进行了系统介绍。上述方法可以实现对海洋药用生物粗提物中常见核苷类化合物的快速筛选、排重,避免对已知成分繁杂的分离纯化、结构鉴定过程,为海洋天然产物化学研究提供了一种新思路。     

高速逆流色谱法分离纯化黄酮类化合物的研究进展

目的综述高速逆流色谱法分离纯化黄酮类化合物的研究进展。方法查阅近年来公开发表的论文、专著等资料,对高速逆流色谱法分离纯化黄酮类化合物进行概述。结果与结论高速逆流色谱法是一种非常有效的分离纯化黄酮类化合物的方法。     

Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling

Three peptide segments corresponding to the complete sequence of the 104 amino acid protein bovine apocytochrome c were synthesized by the solid-phase method. The peptides Ac-[Cys(Cam)^14,17, GlyS²³]-apocytochrome c-(1–23) (I), CF₃CO-[GlyS⁶⁰]-apocytochrome c-(24–60) (II), and CF₃CO-apocytochrome c-(61–104) (III) were purified by chromatography on CM-cellulose, partition chromatography and/or HPLC. Each of the peptides was reacted with citraconic anhydride to block all of the lysine side chains, and the 61–104 peptide was treated with 10% hydrazine to remove the trifluoroacetyl group, to give the corresponding peptides Ia, IIa, and IIIa. Peptides IIa and IIIa were coupled together by reaction with silver nitrate/N-hydroxysuccinimide to give the 24–104 sequence. After removal of the trifluoroacetyl group from the amino terminus, peptide Ia was also coupled. Treatment of the peptide mixture with aqueous acetic acid removed the citraconyl groups, and purification by chromatography on CM-cellulose and HPLC gave a 0.6% yield of [Cys-(Cam)^14,17]-apocytochrome c. The synthetic product was shown to be identical to a sample derived from native bovine cytochrome c by paper or gel electrophoresis, HPLC and by chymotryptic or tryptic map.     

High‐throughput peptide synthesis and peptide purification strategy at the low micromol‐scale using the 96‐well format

Abstract: The increasing demand for short‐ and medium‐sized peptides in many fields of biological, medical and pharmaceutical research requires optimized and universally applicable high‐throughput synthesis and purification techniques at the low‐µmol scale. Here, we describe a continuous peptide synthesis/purification approach using the 96‐well format. First, a µmol scale peptide synthesis on resin beads was optimized on a novel miniaturized 96‐reaction vessel block employing standard Fmoc/tBu‐chemistry. Almost 90% of the synthesized peptides contained the target sequence as the main component, as judged from matrix‐assisted laser desorption/ionization (MALDI) mass spectra. Impurities were mostly related to partially protected peptides. Second, we tested the applicability of ion pair reversed‐phase solid‐phase extraction (IP–RP–SPE) to purify individual peptides. Depending on the length and predicted hydrophobicity of the peptides, elution was performed with 25 or 35% aqueous acetonitrile in the presence of 0.1% trifluoroacetic acid (TFA). Thus, scavengers used during TFA cleavage and partially protected peptides carrying very hydrophobic protecting groups were effectively removed. Using a narrow step gradient, the target peptides were even separated from deleted sequences and protected peptides with similar hydrophobicities. Third, we combined the µmol‐scale synthesis in the 96‐well format with purification by IP–RP–SPE on a 96‐well micro‐extraction plate format. This simple, fast and parallel approach was tested on 12‐mer and 15‐mer peptides to map epitopes of T‐ and B‐cell clones, respectively. Approximately 80% of all peptides were obtained at purities > 90% without purification by RP–HPLC. In summary, this novel approach has several advantages: (i) the µmol‐scale reduced the cost of peptide synthesis, (ii) large numbers of peptides were purified faster, (iii) the volumes of eluents and waste were significantly reduced, and (iv) the RP–HPLC column was not contaminated with hydrophobic impurities.     

pH区带精制逆流色谱在分离生物碱类物质的应用

pH区带精制逆流色谱法是在普通制备型高速逆流色谱分离纯化生物碱类成分的应用基础上发展起来的一种特殊逆流色谱分离制备技术。pH区带精制逆流色谱法由于具备进样量高、分离效率高、分离效果好、杂质易收集、可实现pH监控、待分离组分被高度浓缩等优点,目前已被广泛应用于研究领域与实践中。生物碱类物质是重要的天然产物成分,具有很强的生物活性,对很多疾病有良好的治疗作用。本文介绍了pH区带精制逆流色谱法及其在分离生物碱类天然产物中的应用。