东亚钳蝎4个软瘫型抗昆虫神经毒素的纯化及其分子特征

本文首次直接用反相高效液相色谱(RP-HPLC)对东亚钳蝎(Buthus martensi Karsch)粗毒进行了分离,获得了至今关于该毒神经毒素组分的最佳分离和高回收得率的图谱,如本法可将哺乳动物毒素(包括兼有哺乳动物毒性的昆虫毒素)和只有昆虫毒性的昆虫毒素分成两个区,并从后一区中纯化了多至5个抗昆虫毒素(分别将它们定名为BmK IT1至IT5)。毒性检测、氨基酸组成分析及N-端3肽序列测定结果表明,除其中的BmK IT1应为前文报道的BmK IT(挛缩型抗昆虫毒素)外,BmK IT2至IT5则都是新发现的软瘫型抗昆虫毒素。该4个毒素都是偏碱性的、由约61个氨基酸残基组成的单链多肽,等电点均在8.3至8.5之间。当BmK IT2分子被荧光物质标记修饰后,生物毒性便丧失。揭示这类毒素分子表面带正电荷的残基对维系其生物活性应同样是至关重要的。     

绿豆胰蛋白酶抑制剂片段及其类似物的合成

绿豆胰蛋白酶抑制剂的Lys活性碎片由两条分别含有26及9个氯基酸残基的多肽链通过两对分子间二硫键连接而成。用DTT还原能拆分两链,其中长链含6个半胱氨酸,在空气中氧化后能恢复25％原Lys碎片活力。本文报道了此长链的化学合成和二硫键重组。合成产物的氯基酸组成与文献报道的一致。但活性明显低于天然产物。为此对绿豆抑制剂的部分序列重新进行测定,结果表明原P_2′位的Lys应为Ile按新测定序列,从长链26肽的N端和C端各去掉两个残基合成一个22肽,此22肽的活性与天然的26肽相当。此外还合成了此22肽的类似物,其活性中心的Lys残基由Ala取代,产物对胰蛋白酶和弹性蛋白酶都无抑制活力。     

溴化氰裂解结合电喷雾串联质谱测定电泳分离蛋白的C端序列

C端测序是蛋白质及多肽一级结构确认的重要组成部分,也是重组蛋白药物质量控制的重要依据。建立了溴化氰裂解结合电喷雾串联质谱测定蛋白质C端序列的方法,并应用于重组人肿瘤坏死因子受体和纽兰格林的C端测序。首先根据待测蛋白序列进行溴化氰理论裂解,如果C-端肽段理论分子量在500~5000U之间,则将待测样品进行SDS-PAGE分离,考马斯亮兰染色,然后进行胶内溴化氰裂解,最后应用基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)测定C-端肽段的分子量,电喷雾串联质谱对C端肽段进行测序。应用本方法分别测定了这两个蛋白质C端19个和11个氨基酸残基序列。研究结果表明:本方法灵敏、有效、实用性较强,可适用于部分重组蛋白药物的质量控制和蛋白质的结构确证,是对目前蛋白质C端测序方法的有效补充。     

四棱草环肽C和D的ESIMS-MS分析

利用低能量碰撞诱导解离(CID)技术对四棱草环肽C和四棱草环肽D的电喷雾电离串联质谱(ESIMS-MS)进行了研究.ESIMS可以确定环肽的分子量,根据多级质谱中逐次失去氨基酸残基的碎片离子可以确定环肽中氨基酸残基的连接顺序.氨基酸残基主要从酰氨键C端断裂失去,有时亦会从N端断裂失去氨基酸残基.     

纳升电喷雾串联质谱鉴定重组人甲状旁腺素(1-34)

用先进的纳升电喷雾-四极杆-飞行时间串联质谱鉴定重组人PTH(1-34)。通过ESI-MS测定重组人PTH(1-34)分子量及MS/MS对其胰蛋白酶酶解后的肽段的序列和数据库查询进行结构鉴定。重组PTH(1-34)测定分子量为4115.21,与理论值相比测定相差0.06%。MS/MS测定出其中双电荷离子峰m/z728.4肽段序列为VSEIQLMHNLG,以及其他3个单电荷离子峰的序列。数据库检索后确定重组表达的PTH(1-34)一级结构完全正确,纳升电喷雾串联质谱以其灵敏、快速和准确为蛋白质鉴定提供了有效的手段。     

栝楼蛋白 2: 栝楼蛋白部分化学结构的初步测定

栝楼蛋白(Trichobitacin)是从栝楼(Trichosanthes kirilowiiMaxim, Cucurbitaceae)中新发现的核糖体失活蛋白, 分子量为27,228; pI为9.6。应用基质辅助的激光解析飞行时间质谱(MALDI-TOF-MS)和快原子轰击质谱法(FAB-MS)分别测定胰蛋白酶酶解栝楼蛋白和天花粉蛋白(Trichosanthin)的混合肽质谱, 通过比较发现了一些分子量相同的肽。由于这两种蛋白质都来源于栝楼块根, 同源性比较强, 所以这些肽序列在两种蛋白质中基本一样; 再结合蛋白N-端自动顺序仪测定栝楼蛋白N-端的结果, 确定了栝楼蛋白N-端38个氨基酸的顺序, 栝楼蛋白经胰蛋白酶酶解后所得肽段用HPLC分离纯化, 再用蛋白质自动顺序仪, DABITC/PITC双偶合手工法和质谱法共确定了栝楼蛋白N-端, C-端等100多个氨基酸残基的序列。     

栝楼蛋白 2: 栝楼蛋白部分化学结构的初步测定

栝楼蛋白(Trichobitacin)是从栝楼(Trichosanthes kirilowiiMaxim, Cucurbitaceae)中新发现的核糖体失活蛋白, 分子量为27,228; pI为9.6。应用基质辅助的激光解析飞行时间质谱(MALDI-TOF-MS)和快原子轰击质谱法(FAB-MS)分别测定胰蛋白酶酶解栝楼蛋白和天花粉蛋白(Trichosanthin)的混合肽质谱, 通过比较发现了一些分子量相同的肽。由于这两种蛋白质都来源于栝楼块根, 同源性比较强, 所以这些肽序列在两种蛋白质中基本一样; 再结合蛋白N-端自动顺序仪测定栝楼蛋白N-端的结果, 确定了栝楼蛋白N-端38个氨基酸的顺序, 栝楼蛋白经胰蛋白酶酶解后所得肽段用HPLC分离纯化, 再用蛋白质自动顺序仪, DABITC/PITC双偶合手工法和质谱法共确定了栝楼蛋白N-端, C-端等100多个氨基酸残基的序列。     

肿瘤蛋白D52N-端乙酰化的纳升电喷雾串联质谱分析

小鼠髓系白血病M1细胞经IL-6诱导分化后一系列蛋白质点的表达量发生了变化,其中A点表达量显著增加。MALDI-TOF-MS的肽质量指纹谱鉴定表明A点为肿瘤蛋白D52(tumor protein D52,TP D52)。为进一步确认该结果,对A点进行了ESI-MS/MS分析,测定了3个肽段的序列。Mascot数据库查询结果很肯定地表明该蛋白为TP D52,但只有两个肽段与之匹配。对未匹配肽段的序列分析表明它对应于TP D52N端1-10氨基酸MDRGEQGLLK,其中N端第1个氨基酸甲硫氨酸M发生了乙酰化。分析结果与M乙酰化的规律一致,即当第2个氨基酸是酸性氨基酸D或E时,M很容易发生乙酰化。本研究文首次报道了TP D52的N端乙酰化,其功能需要进一步研究。     

基于精氨酸酶切的蛋白质C端肽段富集方法的优化及评估北大核心CSCD

基于聚合物的蛋白质C端反向富集策略是用于研究蛋白质C端最为广泛的策略之一。目前,基于胰蛋白酶(trypsin)切割精氨酸残基C端(ArgC型酶切)的蛋白C端组学方法对蛋白质C端的鉴定深度仍有待提高。为解决这一问题,该研究对此方法进行了优化和评估:建立了基于“V型”过滤装置的“一锅法”富集流程,避免了副反应的干扰,缩短了样本的制备时间;优化了蛋白水平乙酰化反应条件,最大限度地降低了丝氨酸、苏氨酸、酪氨酸残基上的副反应,提高了肽段鉴定的可信性;优化了基于固相萃取枪头膜片过滤柱(StageTip柱)的样品分离过程,使C端肽段的鉴定深度增加至原来的4倍。通过以上优化,按照肽段水平错误发现率(FDR)<0.01、离子分数(ion score)≥20,且C端带有乙醇胺修饰的数据筛选标准,从人HEK 293T细胞中共鉴定出696个蛋白质C端。若仅要求肽段水平FDR<0.01,鉴定数目进一步增加到933个,这是基于聚合物富集策略的蛋白质C端组学方法所得的最大数据集之一。探索了胰蛋白酶镜像酶(LysargiNase)切割精氨酸残基N端(ArgN型酶切)与不同肽段N端衍生化修饰组合对蛋白质C端鉴定数目和种类的影响,“LysargiNase酶切+肽段N端乙酰化”新策略在原有“胰蛋白酶酶切+肽段N端二甲基化”策略的基础上将鉴定蛋白质C端的种类提升了47%。综上,该研究通过对基于Arg型酶切的蛋白C端组学方法的优化,提升了C端肽段的鉴定深度,扩大了C端肽段鉴定的覆盖范围。该方法将有望成为系统性表征蛋白质C端的有力工具。     

黑麦草叶片超氧化物歧化酶

采用硫酸铵分级沉淀和柱层析方法 ,从黑麦草叶片中分离得到纯的铜锌超氧化物岐化酶。经鉴定该酶是Cu·Zn SOD。测得其分子量约为 3 2 1 0 0 ,亚基分子量约 1 5 90 0。该酶的N 末端为丙氨酸 ,其紫外光区最大吸收峰在 2 5 4nm。该酶的热稳定性较好 ,65℃保温 1h仍保留活性 2 7%。氨基酸组成分析表明 ,黑麦草SOD由 2 1 2个氨基酸残基组成 ,而且不含色氨酸。     

Purification, Characterization and 1H NMR Resonance Assignment of an a－Like Neurotoxin BmK 16 from the Venom of Chinese Scor－pion Buthus martensii Karsch

A natural scorpion toxin BmK 16 was purified for the first time from the venom of the Chinese scorpion Buthus martensii Karsch (BmK) by using combined gel-filtration, ion exchange and reversed phase chromatography. The sequence of the N-terminal 8 amino acid residues was determined by Edman degradation. Using the N-terminal sequence as a tag, the database searching revealed a hit in the scorpion cDNA Bank. The sequence for N-terminal 8 amino acid residues, molecular weight and amino acid compositions of BmK 16 were identical with the calculated values according to the first 64 residues‘ se-quence of the precursor peptide alpha-neurotoxin TX16 derived from the sequence of the cDNA AF156597 (EMBL). The se-quence-specific resonance assignment of BmK 16 was achieved and the intact sequence of BmK 16 was determined as follow-ings: VRDAY IAKPH NCVYE CARNE YCNDL CTKNG AKSGY CQWVG KYGNG CWCKE LPDNV PIRVP GKCH. Furthermore, the results from the sequence homology analysis and the toxicity assays indicated that BmK 16 was an α-likescorpion neurotoxin.     

Purification, characterization and functional expression of a new peptide with an analgesic effect from Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU1)

In this study, a new peptide named BmK AGP‐SYPU1 with an analgesic effect was purified from the venom of Chinese scorpion Buthus martensi Karsch (BmK) through a four‐step chromatographic process. The mouse twisting test was used to identify the target peptides in every separation step. The purified BmK AGP‐SYPU1 was further qualified by RP‐HPLC and HPCE. The molecular mass determined by the MALDI‐4800‐TOF/TOF MS for BmK AGP‐SYPU1 was 7544 Da. Its primary structure of the N‐terminal was obtained using Edman degradation. The gene sequence of BmK AGP‐SYPU1 was cloned from the cDNA pool and genomic of scorpion glands, respectively, and then expressed in Escherichia coli. The sequence determination showed that BmK AGP‐SYPU1 was composed of 66 amino acid residues with a new primary structure. The metal chelating affinity column and cation exchange chromatography were used to purify the recombinant BmK AGP‐SYPU1. Consequently, the native and recombinant BmK AGP‐SYPU1 showed similar analgesic effects on mice as assayed using a mouse twisting model. These results suggested that BmK AGP‐SYPU1 is a new analgesic component found in the Chinese scorpion Buthus martensi Karsch. Copyright © 2010 John Wiley & Sons, Ltd.     

Using accurate mass electrospray ionization-time-of-flight mass spectrometry with in-source collision-induced dissociation to sequence peptide mixtures

Although data-dependent LC-MS-MS with database searching has become au courant for identifying proteins, the technique is constrained by duty-cycle inefficiency and the inability of most tandem mass analyzers to accurately measure peptide product ion masses. In this work, a novel approach is presented for simultaneous peptide fragmentation and accurate mass measurement using in-source collision-induced dissociation (CID) on electrospray ionization (ESI)-time-of-flight (TOF) MS. By employing internal mass reference compounds, mass measurement accuracy within +/-5 ppm for tryptic peptide precursors and +/-10 ppm for most sequence-specific product ions was consistently achieved. Analysis of a complex solution containing several digested protein standards did not adversely affect instrument performance.     

Tandem mass spectrometry of kahalalides: identification of two new cyclic depsipeptides, kahalalide R and S from Elysia grandifolia

Spectra obtained using electrospray ionization mass spectrometry (ESI-MS) of the mollusk Elysia grandifolia showed a cluster of molecular ion peaks centered at a molecular mass of 1478 Da (kahalalide F, an anticancer agent). Two new molecules, kahalalide R (m/z 1464) and S (m/z 1492) were characterized using tandem mass spectrometry. The mass differences of 14 Da suggest that they are homologous molecules. In addition, previously identified kahalalide D and kahalalide G are also reported. However, the ESI-MS of the mollusk's algal diet Bryopsis plumosa showed the presence of only kahalalide F. The amino acid sequences of kahalalide R and S are proposed using collision-induced dissociation (CID) experiments of singly and doubly charged molecular ions and by comparison with the amino acid sequence of kahalalide F. The pathway is presented for the loss of amino acid residues in kahalalide F. It is observed that there is sequential loss of amino acids in the linear peptide chain, but in the cyclic part the ring opens at the amide bond rather than at the lactone linkage, and the loss of amino acid residues is not sequential. The CID experiment of the alkali-metal-cationized molecular ions shows that the sodium and potassium ions coordinate to the amide nitrogen/oxygen in the linear peptide chain of the molecule and not to the lactone oxygen of the lactone. In the case of kahalalide D, CID of the protonated peptide opens the depsipeptide ring to form a linear peptide with acylium ion, and fragment ion signals indicate losses of amino acids in sequential order. In this study, tandem mass spectrometry has provided the detailed information required to fully characterize the new peptides.     

Host-defence skin peptides of the Australian Common Froglet Crinia signifera: sequence determination using positive and negative ion electrospray mass spectra

The amino acid sequences of seven signiferin peptides are provided by consideration of tandem mass spectrometric (MS/MS) data for the respective MH+ and [M--H]- precursor ions. These methods do not differentiate between isomeric residues Leu and Ile; these were identified using the Edman degradation technique. The sequence of signiferin 1, a new smooth muscle contracting neuropeptide (RLCIPYIIPC-OH) containing a disulfide bridge, is best determined using the collision-induced dissociation (CID) spectrum of the [M--H]- anion. The initial fragmentation of this system involves loss of H2S2, which furnishes an open-chain system that is readily sequenced using the alpha and beta backbone cleavage anions.     

Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。     

一种可用于评估肽质量指纹谱数据的方法——反转错位数据库

反转数据库常被用于估算大规模蛋白质组研究中串联质谱搜索数据库结果的可靠性。然而,对于经典的且现在依然在产出的肽质量指纹谱的数据,这种方法并不适用。为解决该问题,构建了另外一种随机数据库,称为反转错位数据库。这种数据库是在反转数据库的基础上,将序列中的K和R及其后的氨基酸交换位置(对于胰蛋白酶切割的结果)获得。这种处理避免了某些肽段因前后胰蛋白酶酶切位点氨基酸相同而在序列反转后质量依然不变,导致肽质量指纹谱法无法区分的问题。通过串联质谱和肽质量指纹谱测试数据的搜索结果,证明了这种方法同时适用于串联质谱和肽质量指纹谱的数据。这种方法扩大了经典反转数据库的适用范围,将对评估和整合串联质谱和肽质量指纹谱的数据具有重要意义。     

Amino Acid Sequence of an Excitatory Insect-selective Toxin (BmK IT) From Venom of the Scorpion Buthus martensi Karsch

The insect-selective neurotoxin(BmK IT) of scorpion Buthus martensi Karsch was first reduced and S-alkylated, and then digested by TPCK-trypsin and Staphylococcus aureus V-8 Protease. The enzymatic peptides were purified on TLC-plastic sheet and submitted to determine their amino acid compositions and sequences. The sequence of the 70 amino acid residues of BmK IT was established with reference to the primary structure of AaH IT, another excitatory insect-selective toxin from the venom of North African scorpion Androctonus australis Hector. About 75% of the homologous sequence was found in the molecules of BmK IT and AaH IT. It is obvious that the results contribute toward better understanding of the molecular structure characteristics, structure/activity relationship of scorpion insect-selective toxins, and they can serve as the molecular basis for utilizing the toxins as a tool to clarify molecular mechanism involved in channel gating, and to infer the possibility of developing them as new selective b     

Low-mass ions produced from peptides by high-energy collision-induced dissociation in tandem mass spectrometry

High-energy collision-induced dissociation (CID) mass spectrometry provides a rapid and sensitive means for determining the primary sequence of peptides. The low-mass region (below mass 300) of a large number of tandem CID spectra of peptides has been analyzed. This mass region contains several types of informative fragment ions, including dipeptide ions, immonium ions, and other related ions. Useful low-mass ions are also present in negative-ion CID spectra. Immonium ions (general structure [H₂N=CH-R]⁺, where R is the amino acid side chain) and related ions characteristic of specific amino acid residues give information as to the presence or absence of these residues in the peptide being analyzed. Tables of observed immonium and reiated ions for the 20 standard amino acids and for a number of modified amino acids are presented. A database consisting of 228 high-energy CID spectra of peptides has been established, and the frequency of occurrence of various ions indicative of specific ammo acid residues has been determined. Two model computer-aided schemes for analysis of the ammo-acid content of unknown peptides have been developed and tested against the database.     

Structural analysis of O-glycopeptides employing negative- and positive-ion multi-stage mass spectra obtained by collision-induced and electron-capture dissociations in linear ion trap time-of-flight mass spectrometry

Structural analyses of various glycans attached to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on mass spectrometry (MS) combining both collision-induced dissociation (CID) and electron-capture dissociation (ECD) in the positive- and negative-ion modes has been proposed as a simple and direct method of assigning an O-glycan without releasing it from the peptide and of determining the amino acid sequence of the peptide and glycosylation site. The instrument used is an electrospray ionization (ESI) linear ion trap (LIT) time-of-flight (TOF) mass spectrometer with tandem LITs for CID by He gas and ECD. The proposed approach was tested with two synthetic O-glycopeptides binding a sialyl Lewis x (sLe(x)) oligosaccharide and a 3'-sialyl N-acetyllactosamine (3'-SLN) on a serine (S) residue. In the negative-ion mode, the CID MS(2) spectra of O-glycopeptides showed a relatively abundant glycoside-bond cleavage between the core N-acetylglucosamine (GlcNAc) and serine (S) that yields deprotonated C(3)-type fragment ions of O-glycan and deprotonated Z(0)-type peptide ions. The structure of the sLe(x) (3'-SLN) oligosaccharide was simply assigned by comparing the CID MS(3) spectrum derived from the C(3)-type fragment ion with the CID MS(2) spectra of the sLe(x) and sLe(a) (3'- and 6'-SLN) standards (i.e., negative-ion MS(n) spectral matching). The amino acid sequence of the peptide including the glycosylation site was determined from the ECD MS(2) spectrum in the positive-ion mode.