序列搜索算法在多肽质谱解析中的应用

序列搜索算法由三部分组成：搜索过程、搜索得到多肽的各氨基酸残基的评分及两端（N端、C端）搜索得到的多肽的合并过程.通过若干实际多肽质谱的解析,结果表明,该算法对多种序列专一性离子并存的未知多肽质谱的解析,可获得较满意结果.尤其是它的评分方式及标准,比较适合多肽质谱图的实际情况,可最大限度地判断解析结果的准确度,为从事用质谱测定多肽一级结构的分析工作者提供了一比较简便且可靠的手段.也为质谱法快速测定蛋白质或多肽序列及其在生物学中的普及提供了一条方便之路.     

用化学方法降解肽链N-末端被封闭的残基及其鉴定

在测定蛋白质或多肽的一级结构时,常会遇到N-末端被封闭的情况,使Edman降解试剂失去作用,无法用正常的降解程序进行蛋白质或多肽的一级结构的测定。N-末端受封闭的种类很多,其中主要是乙(甲)酰化和焦谷氨酰环化。它们大量存在于自然界的蛋白质或活性多肽中,也有部分是在处理过程中通过化学反应形成的。目前对N-末端酰化的肽链结构除了用质谱法等物化方法测定外,几乎没有别的更好的方法,至于焦谷氨酰残基环化的肽     

用化学方法降解肽链N-末端被封闭的残基及其鉴定

在测定蛋白质或多肽的一级结构时,常会遇到N-末端被封闭的情况,使Edman降解试剂失去作用,无法用正常的降解程序进行蛋白质或多肽的一级结构的测定.N-末端受封闭的种类很多,其中主要是乙(甲)酰化和焦谷氨酰环化.它们大量存在于自然界的蛋白质或活性多肽中,也有部分是在处理过程中通过化学反应形成的.目前对N-末端酰化的肽链结构除了用质谱法等物化方法测定外,几乎没有别的更好的方法,至于焦谷氨酰残基环化的肽     

荧光胺的合成和应用

近几年来,在蛋白质化学研究中,分析微量化的重要方面之一是使用荧光方法。在氨基酸、多肽、蛋白质测定中,荧光胺(Fluorescamine)可与游离的氨基反应,产生荧光的物质,借此测定的灵敏度可提高达10~(-12) 克分子。荧光胺在柱层析、薄层层析中都可用来测定氨基酸和多肽;也有用作抗体荧光标记的荧光试剂。此试剂已有Hoffman-La Roche公司生产出售,由于此试剂价格昂贵,因而很多实验室都不能普遍使用。本实验室使用国内的现有试剂,参照了有关的方法尝试合成该试剂,现已初步合成。合成的试剂与进口的Hoffman-La Roche公司的产品作了比较,基本符合。并且就此试剂在溶液     

人表皮生长因子肽谱及一级结构的质谱法分析

采用液相色谱－质谱联用法（ＬＣ－ＭＳ）,分别对基因工程表达的人表皮生长因子胰蛋白酶和Ｖ８蛋白酶的酶解产物进行了肽质谱分析,获得各肽段的分子量信息。并对大部分肽段用串联质谱（ＭＳ－ＭＳ）分析了肽的序列,获得其氨基酸序列信息。此外,还测定了分子中二硫键的数目。均获得满意的结果。这一方法的建立将有助于天然的,人工合成的及基因工程表达的蛋白质或多肽的鉴定和结构分析,其精确度及分辨率都超过常规方法。     

反相色谱在重组多肽精细纯化中的应用

反相色谱因其高分辨率在蛋白质、多肽及其他有机分子的高效液相色谱分析中被广泛使用,特别在基因工程多肽类药物的精细化中起着难以替代的作用。采用反相色谱进行两种重组多肽的精细纯化,获得了好的分离效果。     

蛋白质C端(异)硫氰酸法序列测定反应机理及进展

蛋白质C端测序是蛋白质化学与分子生物学中一门有重要意义的技术,由Schlack和Kumpf于1926年提出的(异)硫氰酸法在该领域发展迅速并占主导地位.对(异)硫氰酸法的反应机理,最新进展以及标准乙内酰硫脲氨基酸制备进行了较详尽的介绍.     

生物学文摘

我国首座“自动蛋白质化学工作站”由湖南师大推出湖南师范大学生物研究所蛋白质化学及分子生物学重点实验室主任梁宋平博士领导的课题组,经过近4年的努力,研制成功我国第一台由计算机自动控制、能进行蛋白质顺序测定和多肽合成及其它蛋白质化学实验的“自动蛋白质化学工作站”。最近,湖南省科委组织了由生物化学专家组成的鉴定委员会,对该工作站进行了现场测试和鉴定。该工作站由微机程控、能自动完成蛋白质化学N端测识、c端测序和多肽合成,该工作站对我国生命科学的研究将起到积极推动作用。（摘自《中国科学报n997．05．02）手术中…     

辅助噬菌体在噬菌体展示中的应用及研究进展

噬菌体展示技术是一种将外源肽或蛋白质与特定噬菌体衣壳蛋白相融合,展示于噬菌体表面来构建蛋白质或多肽文库,并从中筛选目的蛋白、多肽或抗体的基因工程高新技术。噬菌粒/辅助噬菌体系统是最常用的噬菌体展示系统,此系统中辅助噬菌体对噬菌粒的复制和组装发挥着至关重要的作用。本文结合当今该领域的最新研究动态,概述了噬菌粒和辅助噬菌体双基因组系统,着重介绍了不同辅助噬菌体的特点及其突变机制,并对其应用前景进行了展望,以期为该技术的进一步完善提供一定的借鉴作用。     

菠菜和黄瓜光系统Ⅱ捕光叶绿素a／b蛋白质复合体的比较研究

用菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅａ Ｍｉｌｌ．）和黄瓜（Ｃｕｃｕｍ ｉｓｓａｔｉｖｕｓＬ．）叶片的叶绿体制备出光系统Ⅱ捕光叶绿素ａ／ｂ 蛋白质复合体（ＬＨＣⅡ）,并对这两种ＬＨＣⅡ的聚合状态的Ｃｈｌａ／ｂ 值、光谱特性以及多肽组分进行了比较研究。实验结果表明,菠菜ＬＨＣⅡ的Ｃｈｌａ／ｂ 为１．３３,黄瓜ＬＨＣⅡ的Ｃｈｌａ／ｂ 为１．１７。其光谱特性说明黄瓜的ＬＨＣⅡ更富含Ｃｈｌｂ。它们的多肽组分存在着明显差异,菠菜的ＬＨＣⅡ含有２７ ｋＤ和２５ ｋＤ ２个多肽,而黄瓜的ＬＨＣⅡ只含有２７ ｋＤ １个多肽,这表明２５ ｋＤ多肽含有较少的Ｃｈｌｂ。叶绿素蛋白质复合体的分析结果表明,菠菜ＬＨＣⅡ的单体、二聚体及三聚体均由２个多肽组成;而黄瓜的ＬＨＣⅡ不同聚合状态均由１个多肽组成     

Recombinant protein purification using complementary peptides as affinity tags

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.     

The amino acid sequence of avian thymic hormone, a parvalbumin

The complete amino acid sequence of 'avian thymic hormone' (ATH), a protein from thymus tissue that appears to promote immune maturation in chicken bone marrow cells in culture, is presented. The sequence was obtained from sequences of ATH peptides isolated by HPLC after tryptic, chymotryptic, peptic or S aureus V8 protease digestions. The protein is a parvalbumin consisting of 108 residues with a blocked amino terminus, a single cysteine, tyrosine, proline and arginine and no histidine, methionine or tryptophan. This is the first amino acid sequence of a parvalbumin which is not derived from muscle tissue.     

Sequencing of peptides and proteins from the carboxy terminus.

A new chemical method for carboxy-terminal (C-terminal) protein sequencing has been developed. This approach has been successfully used to sequence 5 residues of standard proteins and 5 to 10 residues of synthetic peptides at low nanomole levels. The sequencing procedure consists of converting the C-terminal amino acid into a thiohydantoin (TH) derivative, followed by transformation of the TH into a good leaving group by alkylation. Next, the alkylated TH is cleaved mildly and efficiently with (N = C V S)- anion, which simultaneously forms a TH on the newly truncated protein or peptide. Thus, after the initial TH derivatization, there is no return to a free carboxyl group at the C-terminus. An additional benefit of this method is that the alkylating moiety can be chosen with a variety of properties allowing for variation in the detection method. This chemistry has been adapted to automated protein sequencers with a cycle time of about 1 h.     

Noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the alpha-helix have the same helical propensity

It was established previously that helical propensities of different amino acid residues in the middle of α‐helix in peptides and in proteins are very similar. The statistical analysis of the protein helices from the known three‐dimensional structures shows no difference in the frequency of noncharged residues in the middle and at the C terminus. Yet, experimental studies show distinctive differences for the helical propensities of noncharged residues in the middle and in the C terminus in model peptides. Is this a general effect, and is it applicable to protein helices or is it specific to the model alanine‐based peptides? To answer this question, the effects of substitutions at positions 28 (middle residue) and 32 (C2 position at the C terminus) of the α‐helix of ubiquitin on the stability of this protein are measured by using differential scanning calorimetry. The two data sets produce similar values for intrinsic helix propensity, leading to a conclusion that noncharged amino acid residues at the solvent‐exposed positions in the middle and at the C terminus of the α‐helix have the same helical propensity. This conclusion is further supported with an excellent correlation between the helix propensity scale obtained for the two positions in ubiquitin with the experimental helix propensity scale established previously and with the statistical distribution of the residues in protein helices.     

Novel thioester reagents afford efficient and specific S-acylation of unprotected peptides under mild conditions in aqueous solution.

S-acylated peptides have many potential uses for elucidating the biophysical, structural and other properties of the numerous S-acylated proteins of mammalian cells. However, with the currently available reagents, preparation of specifically S-acylated derivatives of peptides is generally laborious or simply unfeasible. We here show that novel, easily preparable aryl and alkyl thioester derivatives of palmitic acid can mediate S-acylation of peptides corresponding to physiologically S-acylated sequences from the proteins p56(lck) and H-ras and the Po glycoprotein of peripheral myelin, with high selectivity for cysteine over other amino acid functional groups (including hydroxyl and both alpha- and epsilon-amino residues), and with much greater efficiency than is obtained using acyl-coenzyme A derivatives. Efficient and selective S-acylation can be accomplished under very mild conditions in aqueous systems containing lipid vesicles or detergent micelles, or in homogenous aqueous/acetonitrile mixtures. Using these novel thioesterifying reagents, we confirm previous suggestions that the N-terminal cysteine residue of Hedgehog proteins can exhibit rapid, uncatalyzed S-to-N acyl transfer following S-acylation to produce the N-palmitoylated amino terminus found in the mature protein. By contrast, we demonstrate that spontaneous S-to-N acyl transfer from the cysteine to the terminal glycine residue in the amino-terminal peptide of G(alphas) is far less rapid and is likely too slow to explain the physiological N-palmitoylation of the amino terminus of this protein.     

Synthesis and characterization of insulin-like growth factor (IGF)-1 photoprobes selective for the IGF-binding proteins (IGFBPS). photoaffinity labeling of the IGF-binding domain on IGFBP-2

Elevated insulin-like growth factor (IGF)-1 levels are prognostic for the development of prostate and breast cancers and exacerbate the complications of diabetes. In each case, perturbation of the balance between IGF-1/2, the IGF-1 receptor, and the IGF-binding proteins (IGFBPs) leads to elevated IGF-1 sensitivity. Blockade of IGF action in these diseases would be clinically significant. Unfortunately, effective IGF antagonists are currently unavailable. The IGFBPs exhibit high affinity and specificity for the IGFs and serve as natural IGF antagonists, limiting their mitogenic/anti-apoptotic effects. As an initial step in designing IGFBP-based agents that antagonize IGF action, we have begun to analyze the structure of the IGF-binding site on IGFBP-2. To this end, two IGF-1 photoprobes, N(alphaGly1)-(4-azidobenzoyl)-IGF-1 (abG(1)IGF-1) and N(alphaGly1)-([2-6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3'-dithiopropionoyl)-IGF-1 (bedG(1)IGF-1), selective for the IGFBPs were synthesized by derivatization of the alpha-amino group of Gly(1), known to be part of the IGFBP-binding domain. Mass spectrometric analysis of the reduced, alkylated, and trypsin-digested abG(1)IGF-1.recombinant human IGFBP-2 (rhIGFBP-2) complex indicated photoincorporation near the carboxyl terminus of rhIGFBP-2, between residues 266 and 287. Mass spectrometric analysis of avidin-purified tryptic peptides of the bedG(1)IGF-1.rhIGFBP-2 complex revealed photoincorporation within residues 212-227. Taken together, these data indicate that the IGFBP-binding domain on IGF-1 contacts the distal third of IGFBP-2, providing evidence that the IGF-1-binding domain is located within the C terminus of IGFBP-2.     

Alpha helix capping in synthetic model peptides by reciprocal side chain–main chain interactions: Evidence for an N terminal “capping box”

A significant fraction of the amino acids in proteins are alpha helical in conformation. Alpha helices in globular proteins are short, with an average length of about twelve residues, so that residues at the ends of helices make up an important fraction of all helical residues. In the middle of a helix, H-bonds connect the NH and CO groups of each residue to partners four residues along the chain. At the ends of a helix, the H-bond potential of the main chain remains unfulfilled, and helix capping interactions involving bonds from polar side chains to the NH or CO of the backbone have been proposed and detected. In a study of synthetic helical peptides, we have found that the sequence Ser-Glu-Asp-Glu stabilizes the alpha helix in a series of helical peptides with consensus sequences. Following the report by Harper and Rose, which identifies SerXaaXaaGlu as a member of a class of common motifs at the N termini of alpha helices in proteins that they refer to as “capping boxes,” we have reexamined the side chain–main chain interactions in a varient sequence using ¹H NMR, and find that the postulated reciprocal side chain-backbone bonding between the first Ser and last Glu side chains and their peptide NH partners can be resolved: Deletion of two residues N terminal to the Ser-Glu-Asp-Glu sequence in these peptides has no effect on the initiation of helical structure, as defined by two-dimensional (2D) NMR experiments on this variant. Thus the capping box sequence Ser-Glu-Asp-Glu inhibits N terminal fraying of the N terminus of alpha helix in these peptides, and shows the side chain–main chain interactions proposed by Harper and Rose. It thus acts as a helix initiating signal. Since normal a helix cannot propagate beyond the N terminus of this structure, the box acts as a termination signal in this direction as well. © 1994 John Wiley & Sons, Inc.     

Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.     

一种优化的MALDI-TOF质谱分析多肽C端序列方法

利用基质辅助激光解吸飞行时间 (MALDI TOF)质谱技术 ,测定羧肽酶Y消化蛋白质和多肽 .所产生的缩短肽片段的质量 ,在一张谱图上得到各个不同酶解时间所形成的肽质量梯度 .根据谱图中相邻两肽峰的质量差得到切去氨基酸的信息 ,从而读出C端氨基酸序列 .在pmol水平下对人促肾上腺皮质激素片段 (ACTH 1 3 9) ,人血管紧张肽片段 (angiotensin Ⅰ ,angiotensin Ⅱ )的C端序列进行了测定 .讨论了在不同浓度 ,不同时间 ,不同温度下酶解所得到的序列测定结果 .在优化条件下 ,人ACTH片段得到了C端 2 0个氨基酸残基顺序 ,为目前C端序列分析所得到的最长序列     

Motion of carboxyl terminus of Galpha is restricted upon G protein activation. A solution NMR study using semisynthetic Galpha subunits

The carboxyl terminus of the G protein alpha subunit plays a key role in interactions with G protein-coupled receptors. Previous studies that have incorporated covalently attached probes have demonstrated that the carboxyl terminus undergoes conformational changes upon G protein activation. To examine the conformational changes that occur at the carboxyl terminus of Galpha subunits upon G protein activation in a more native system, we generated a semisynthetic Galpha subunit, site-specifically labeled in its carboxyl terminus with 13C amino acids. Using expressed protein ligation, 9-mer peptides were ligated to recombinant Galpha(i1) subunits lacking the corresponding carboxyl-terminal residues. In a receptor-G protein reconstitution assay, the truncated Galpha(i1) subunit could not be activated by receptor; whereas the semisynthetic protein demonstrated functionality that was comparable with recombinant Galpha(i1). To study the conformation of the carboxyl terminus of the semisynthetic G protein, we applied high resolution solution NMR to Galpha subunits containing 13C labels at the corresponding sites in Galpha(i1): Leu-348 (uniform), Gly-352 (alpha carbon), and Phe-354 (ring). In the GDP-bound state, the spectra of the ligated carboxyl terminus appeared similar to the spectra obtained for 13C-labeled free peptide. Upon titration with increasing concentrations of AlF4-, the 13C resonances demonstrated a marked loss of signal intensity in the semisynthetic Galpha subunit but not in free peptide subjected to the same conditions. Because AlF4- complexes with GDP to stabilize an activated state of the Galpha subunit, these results suggest that the Galpha carboxyl terminus is highly mobile in its GDP-bound state but adopts an ordered conformation upon activation by AlF4-.