重组人纽兰格林二硫键分析

目的：应用多种质谱技术确证重组人纽兰格林（rhNRGL）的一级结构及其二硫键定位。方法：（1）利用Q—FT—MS测定rhNRGL二硫键还原前后的精确相对分子质量，确定分子中二硫键数目；（2）应用MALDI—TOF／TOF—MS法测定rhNRGL的N端序列；（3）用ESI-MS／MS法测定rhNRGL的C端序列；（4）通过Trypsin和Glu—C2种蛋白酶进行酶解，获得肽质量指纹谱，确证其序列及定位3对二硫键。结果：（1）测定rhNRGL还原前后的精确相对分子质量，两者相差6．0516，确证其主成分形成了3对二硫键；（2）串联质谱法测定rhNRGL的N端序列的5个氨基酸和C端序列的11个氨基酸，均与理论序列一致；（3）2种酶切肽谱的序列覆盖率分别为82％和64％，分析确证主成分二硫键配对正确，但同时存在少量错配二硫键异构体。结论：以上结果表明，该样品的一级结构正确，主要二硫键模式为：1—3，2—4，5—6。多肽药物的二硫键分析常常需要多种质谱及样品制备技术联合。     

奥曲肽一级结构的确证

目的：对奥曲肽(人工合成8肽)的一级结构进行确证。方法：采用：HPLC法测定了奥曲肽的氨基酸组成，采用：Edman降解法测定了除苏氨醇外的7个氨基酸的序列，采用ESI—MS／MS对奥曲肽的分子量以及C—末端的苏氨醇残基进行了确证。结果：采用HPLC法测得除色氨酸以外，样品的氨基酸组成与理论值基本一致。采用Edman降解法测得除苏氨醇外其余7个氨基酸序列与理论结构一致。采用ESI—MS／MS测得样品的相对分子质量为1018．7，与理论值1019．3基本符合。对二级质谱碎片的解析确证样品C—末端确为苏氨醇残基。结论：本文对奥曲肽的C—末端苏氨醇残基以及一级结构全序列进行了确证。     

电喷雾-四极杆-飞行时间串联质谱(ESI-Q-TOF2)鉴定重组人FKBP12

目的用最新的生物质谱技术-电喷雾-四极杆-飞行时间串联质谱(ESI-Q-TOF2)鉴定重组蛋白质rhFKBP12.方法通过ESI-MS测定rhFKBP12分子量及ESI-MS/MS测定其胰蛋白酶酶切后肽段的序列和数据库查寻进行结构鉴定.结果 rhFKBP12的测定分子量为11 820.38,与理论值相比测定误差为0.007%.ESI-MS/MS测定出的两个肽段的部分序列分别为QVETMS和EEGVAQMSV,用这两段序列查寻数据库的结果表明rhFKBP12的结构正确.结论 ESI-MS/MS是鉴定蛋白质的灵敏、快速和准确的新方法.     

重组人甲状旁腺素1—34肽谱及一级结构的液相色谱／电喷雾离子化质谱法分析

目的：采用液相色谱／电喷雾质谱联用法(LC—ESI—MS／MS)对重组人甲状旁腺素1—34(rh—PTH—34)进行肽谱及一级结构分析。方法：用LC—ESI—MS法测定相对分子质量，并在线分析其胰蛋白酶和V8蛋白酶酶解质量肽谱；结合质谱源内碰撞诱导电离技术解析酶解肽段的氨基酸序列。结果：相对分子质量测定值与理论值一致；胰蛋白酶和V8蛋白酶酶解肽段氨基酸序列与理论值基本一致，序列覆盖率分别为97％和88％。结论：LC—ESI—MS／MS联用法为基因重组多肽类药物的一级结构分析和质量控制提供了新途径。     

LC-MS/MS法对融合蛋白FP3的氨基酸全序列测定

运用液质联用、两种串联质谱对融合蛋白FP3的氨基酸全序列测定, 确证其一级结构。将样品还原烷基化后, 通过胰蛋白酶酶解蛋白, PNGase F去除多肽混合物中糖肽的糖基化, 将去糖后的总肽用于液质联用系统, 通过液相分离后, 运用Q-TOF和线性离子阱两种串联质谱测定各个肽段的b, y碎片离子, 分析测定融合蛋白FP3的氨基酸全序列。通过LC-ESI-Q-TOF完成了融合蛋白FP3的76%氨基酸序列测定, 通过LC-ESI-Trap完成余下24%氨基酸序列测定。液质联用、串联质谱法测定蛋白质氨基酸序列快速、灵敏、准确, 是对重组蛋白结构分析和确证的重要手段。     

液质联用分析重组人白细胞介素-11的肽图

目的用液质联用技术分析鉴定重组人白细胞介素-11(rhIL-11)的肽图。方法用胰蛋白酶酶解rhIL-11，采用HPLC测定肽图，用电喷雾-四极杆-飞行时间质谱(ESI-Q-TOF/MS)技术分析肽段的精确相对分子质量，通过串联质谱(MS/MS)测定肽段的氨基酸序列。结果根据肽段的色谱保留时间、相对分子质量及对其碰撞诱导解离质谱的解析结果，归属出肽图中各肽段所在的色谱峰，已检出肽段的总覆盖率为97%。结论液质联用研究肽图是验证和分析蛋白质结构的高效准确的方法。     

应用基质辅助激光解析电离飞行时间质谱和N端测序鉴定重组人粒／巨噬细胞集落刺激因子（rhGM—CSF）的结构

目的：应用基质辅助激光解析电离飞行时间质谱（MALDI－TOF－MS）和N端测序方法鉴定重组人粒／巨噬细胞集落刺激因子（rhGM-CSF)的结构。方法：1．用Edman降解法进行N端测序。2．应用MALDI－TOF－MS测定分子量并鉴定纯度。3．通过还原、烷基化确定rhGM-CSF分子中二硫键数目。4．通过蛋白内切酶Glu-C酶切的肽质量指纹谱确证氨基酸序列并确证其中两对二硫键的配对。结果：1．测定的rhGM-CSF N端15个氨基酸序列与从cDNA推导的序列一致。2．测定的rhGM-CSF分子量与理论计算值一致。3．证明rhGM-CSF没有自由巯基,含有两对二硫键,与理论值一致。4．证明rhGM-CSF的氨基酸序列是正确的,并确证其中的两对二硫键配对正确。结论：确证重组人粒／巨噬细胞集落刺激因子（rhGM-CSF)的结构是正确的,没有修饰、变异和氨基酸的丢失。应用的方法灵敏、准确、可靠。     

纳升电喷雾串联质谱在修饰多肽及未知多肽结构分析中的应用

目的：以2种多肽为例，说明纳升电喷雾串联质谱存修饰多肽及未知多肽结构一级结构确证和分析中的应用。方法：存纳升电喷雾串联质谱TOF MS模式下对2种多肽的分子量进行了确认，然后在MS／MS模式下获得双电倚肽段的碎片离子峰。结果：曲谱瑞林一级结构全序列是E’HWSYWLRPG’，其中N-端焦谷氨酸和C-端甘氨酰胺。未知多肽通过“序列拼接法”得到该样品的全序列T’VSP^＊VWLPPSVY，其中第4位脯氨酸加合了一个钠离子，且N-端苏氨酸磷酸化。结论：本研究表明纳升电喷雾串联质谱在分析修饰多肽及未知多肽方面具有明显优势。     

基于液质联用技术的溶菌酶一级结构分析研究

目的:使用超高效液相色谱-四极杆串联飞行时间质谱法(UPLC-Q TOF MS法)对国内外3家企业溶菌酶的一级结构进行分析研究。方法:采用BEH300 C₄(50 mm×2.1 mm, 1.7μm)色谱柱和BEH300 C₁₈(150 mm×2.1 mm, 1.7μm)色谱柱，以0.1%甲酸/水溶液为流动相A,0.1%甲酸/乙腈溶液为流动相B,梯度洗脱，流速0.2 mL·min^-1;采用Q TOF MS的电喷雾正离子模式，相对分子质量测定时采用MS模式进行扫描，氨基酸序列和二硫键连接方式测定时采用MS^E模式进行扫描。结果:3种来源溶菌酶相对分子质量与理论值一致；肽图结果显示各样品的氨基酸序列均与理论一致；二硫键连接方式均为Cys6-Cys127、Cys30-Cys115、Cys64-Cys80、Cys76-Cys94。结论:UPLC-Q TOF MS法可用于溶菌酶一级结构的分析。     

MALDI-TOF-MS在奥曲肽结构分析中的应用

目的:应用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对奥曲肽进行结构确证分析。方法:采用二巯基苏糖醇还原奥曲肽二硫键,碘代乙酰胺(IAM)封闭还原奥曲肽中的巯基,测定还原前后及烷基化奥曲肽相对分子质量,并应用源后衰变(PSD)对其进行序列分析。结果:奥曲肽、还原奥曲肽和烷基化奥曲肽相对分子质量测定值分别为1019.25,1021.24,1135.38,与 PSD 测定的序列相同,均和理论值一致。结论:本方法可以较快速分析奥曲肽结构,是此类多肽结构确证的有效方法。     

重组L-天门冬酰胺酶II的液相色谱/电喷雾离子化质谱法分析

目的　对基因重组L-天门冬酰胺酶II产品进行一级结构分析。方法　采用流动注射方式,电喷雾离子化质谱法测定分子量;三氯乙酸变性,胰蛋白酶水解后,HPLC测定肽图谱;结合质谱源内碰撞诱导解离技术,解析酶解肽段的氨基酸顺序。结果　分子量测定值与理论值不符,质谱解析表明样品中存在3个氨基酸变异现象,由此计算的分子量与测定值吻合。结论　液相色谱/电喷雾质谱法为基因重组蛋白质药物的一级结构分析和质量控制提供了新途径。     

Propeptin,a new inhibitor of prolyl endopeptidase produced by microbispora II. Determination of chemical structure

The structure of propeptin, a new inhibitor of prolyl endopeptidase isolated from Microbispora sp. SNA-115, was determined. FAB/MS, Edman degradation and amino acid analysis revealed propeptin to be a cyclic polypeptide consisting of 19 common L-amino acids. By FAB/MS and protein chemical methods, the primary sequence of propeptin was determined to be Gly1-Tyr-Pro-Trp-Trp-Asp-Tyr-Arg-Asp9-Leu-Phe-Gly-Gly-His-Thr-Phe-Ile-Ser-Pro19, which cyclizes between the beta-carboxyl group of Asp9 and the a-amino group of Gly1.     

Structure determination of neoefrapeptins A to N: peptides with insecticidal activity produced by the fungus Geotrichum candidum

The structures of neoefrapeptins A to N, peptides with insecticidal activity, were elucidated. They showed a close similarity to efrapeptin. However, all neoefrapeptins contained the very rare amino acid 1-amino-cyclopropane-carboxylic acid and some of them also contained (2S,3S)-3-methylproline. The neoefrapeptins are the first case, in which these amino acids are found as building blocks for linear peptides. They were identified by comparison of the silylated hydrolyzate to reference material by GC/MS (EI-mode). The sequence was elucidated using mass spectrometry (ESI+ mode). Full scan spectra showed two fragments in high yield, even under mild ionization conditions. MS/MS spectra of these two fragments yielded fragment rich spectra from which the sequence of the compounds was determined almost completely. The proteolytic cleavage with the proteinase papain yielded products that allowed to prove the rest of the sequence and the identity of the C-terminus to efrapeptin. The proteolytic cleavage products allowed furthermore to determine the position of the isobaric amino acids, pipecolic acid and 3-methylproline in neoefrapeptin F, as well as the location of R-isovaline and S-isovaline. Papain digestion was such established as a tool for structure elucidation of peptides rich in alpha,alpha-dialkylated amino acids. CD spectra suggested a 3(10) helical structure for neoefrapeptins A and F.     

SPF-5506-A4, a new peptaibol inhibitor of amyloid beta-peptide formation produced by Trichoderma sp

A new peptaibol compound, SPF-5506-A4, was isolated from the fermentation broth of Trichoderma sp. SPF-5506. The chemical structure of the 14-residue peptide was determined by MS, NMR and amino acid sequence analyses. The absolute configuration of amino acid residues in the acid hydrolysate was determined by Marfey's method. The structure of SPF-5506-A4 was established as Ac-Aib-L-Asn-L-Ile-Aib-L-Pro-L-Ser-L-Ile-Aib-L-Pro-L-Leu-L-Leu-Aib-L-Pro-L-leucinol. The compound inhibited amyloid beta-peptide formation in primary guinea pig cerebral cortex neuron cell culture dose-dependently with an IC50 of 0.1 microg/ml. Cytotoxicity was not observed at concentrations of <3 microg/ml.     

The isolation and characterization of a peptide that alters sodium channels from Buthus martensii Karsch.

The peptides were purified using gel filtration, ion exchange, FPLC, and HPLC chromatography and found to greatly prolong action potentials at nanomolar concentrations when applied to frog and mouse nerves. The N-terminal primary amino acid sequence of one of the peptides, BMK 16(5), was determined. The first 23 amino acids of BMK 16(5) were found to be: VKDGYIADDRNCPYFCGRNAYYD. The two cysteine residues in the sequence appeared as Edman sequence cycle blanks; however, they were assigned to be cysteines due to sequence similarity to other peptide toxins that bind to sodium channels and identification of the presence of cysteines obtained from single time point amino acid analysis. The MW of BMK 16(5) was determined by a Perkin Elmer API 300 LC/MS/MS to be 3,695. The amino acid residues of BMK 16(5) show strong similarity with the first 23 amino acid residues of a number of scorpion alpha neurotoxins. Unlike these neurotoxins, BMK 16(5) possesses a proline residue at position 13 which will likely make it fold in a unique way so as to bind to and alter sodium channels.     

A new anti-MRSA antibiotic complex, WAP-8294A II. Structure characterization of minor components by ESI LCMS and MS/MS

The anti-MRSA antibiotic, WAP-8294A, was isolated from the fermentation broth of Lysobacter sp. The major component, WAP-8294A2, is composed of 1 mol of Gly, L-Leu, L-Glu, D-Asn, D-Trp, D-threo-β-hydroxyasparagine, N-Me-D-Phe and N-Me-L-Val, and 2?mol of L-Ser, D-Orn and D-3-hydroxy-7-Me-octanoic acid. The structure of the WAP-8294A2 was mainly determined as a cyclic depsipeptide by 2D NMR experiments. However, it was difficult to use the NMR experiment to determine the minor components, A1, A4 and Ax13, isolated in small amounts. In the present study, ESI MS/MS was applied to the structure elucidation of these minor components. The structures of these minor components were determined on the basis of the fragmentation pattern of the product ions of WAP-8294A2 in the ESI MS/MS. As a result, it was confirmed that A1 and A4 had the same amino acid sequence as A2, while A1 and A4 had the 3-OH-octanoic acid and 3-OH-8-Me-nonanoic acid, respectively, in the place of the 3-OH-7-Me-octanoic acid in A2. In the structure of Ax13, it was found that Gly of A2 was changed to β-Ala of Ax13.     

Goadsporin, a chemical substance which promotes secondary metabolism and Morphogenesis in streptomycetes. II. Structure determination

The structure of goadsporin was determined by using spectroscopic techniques. NMR analysis revealed that goadsporin consists of 19 amino acids, two of which are dehydroalanines (Deala), and six of which are cyclized to oxazoles (Oxz) and thiazoles (Thz) by dehydrative cyclization and dehydrogenation from serine, threonine and cysteine. NMR analysis established seven partial structures, and their sequence was determined by CID-MS/MS. Negative mode FAB-MS/MS gave product ions arising from charge-remote fragmentation that allowed determination of the sequence of the amino acid components as AcNH-Ala-MeOxz-Val-Deala-MeOxz-Ile-Leu-Thz-Ser-Gly-Gly-MeOxz-Leu-Deala-Oxz-Ala-Gly-Thz-Val-OH. The chiral amino acids were determined by the advanced Marfey's method to have L-configurations.