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。     

液质联用鉴定重组人血管内皮抑制素

目的：用液质联用技术鉴定重组人血管内皮抑制素（rhEndostatin）。方法：利用ESI—Q—TOF—MS法测定rhEndostatin的精确相对分子质量，通过HPLC—ESI—Q—TOF—MS／MS法测定其胰蛋白酶酶切后肽段的部分氨基酸序列并结合数据库检索进行结构鉴定。结果：重组人血管内皮抑制素的实测相对分子质量为21114．5，与理论相对分子质量21113．8相比非常接近。HPLC—ESI—Q—TOF—MS／MS测定m／z802．0的肽段的部分氨基酸序列为Ala—Pro—Ser—Ala-Thr—Gly—Gin—Ala—Ser—Ser—Leu—Leu。将其m／z和氨基酸序列在MASCOT数据库检索，结果表明重组人血管内皮抑制素的结构正确。结论：液质联用是鉴定蛋白质的灵敏、快速、准确的新方法。     

白细胞介素－2的一级结构分析

为了鉴定白细胞介素－2的一级结构序列，通过对IL－2样品进行胰酶酶切，将IL－2蛋白分裂成多个小分子肽段，再进行高效液相色谱分离，收集各肽段片段。然后通过MALDI－TOF－MS一级解析鉴定各肽段的分子量，并对各肽段进行二次解析，鉴定各肽段的氨基酸序列。通过以上方法鉴定了IL－2样品的一级结构，并对二硫键进行了定位，证明了该方法的可行性。对该方法的进行的初步应用研究，为 IL－2蛋白的检测方法建立提供了依据。     

依替巴肽一级结构的确证

目的:对合成的依替巴肽一级结构进行确证。方法:采用串联质谱序列分析法测定了氨基酸序列,采用 ESI-MS/MS 对依替巴肽的相对分子质量和肽链环化结构进行了确证,采用~1 HNMR 进一步确定了依替巴肽的结构。结果:串联质谱序列分析法,证实合成肽氨基酸序列和非天然氨基酸结构正确;ESI-MS/MS 测得样品准确的相对分子质量为831.48,与依替巴肽的参照品及理论值(831.96)完全一致;质谱分析确证合成依替巴肽分子内形成了一个正确的二硫键;~1 HNMR 进一步确定了依替巴肽分子的一级结构。结论:本文对依替巴肽的氨基酸序列以及一级结构全序列进行了确证。     

重组人粒细胞巨噬细胞集落刺激因子效价测定中最佳细胞密度的探讨

重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF)效价的测定是采用TF-1rhGM-CSF依赖株细胞,该细胞的增殖与rhGM-CSF浓度成正比,用MTT法测定细胞增殖程度,反映rhGM-CSF的活性,在测定rhGM-CSF的效价过程中TF-1细胞的密度对实验结果有直接影响。     

重组人粒细胞-巨噬细胞集落刺激因子溶液中的稳定性初探

在大肠杆菌表达的重组人粒细胞-巨噬细胞集落刺激因子（rhGM-CSF）纯化的基础上,我们研究了其在溶液中的稳定性,发现它在pH6.8的PBS缓冲液中稳定性很好,但在pH8.3的Tris·HCl冲浪中时隔2 wk以上即发生降解,SDS-BAGE分析,相应位置变成两条带,将SDS-PAGE胶电转移至PVDF膜,将膜上出现的两条蛋白染色带剪下,进行N端氨基酸序列分伍,两蛋白均有固定的降解位点.降解的蛋白与未降解的相比,生物活性仅有较激下降。     

基质辅助激光解析电离飞行时间质谱用于产碳青霉烯酶肺炎克雷伯菌ST11分型的研究

目的：探讨基质辅助激光解析电离飞行时间质谱（ MALDI?TOF MS）用于产碳青霉烯酶肺炎克雷伯菌ST11分型。方法收集2012年10月－2014年10月5所医院的80株产碳青霉烯酶肺炎克雷伯菌菌株。经多位点序列分析（ MLST）,28株为ST11型,52株为其他分型（ other sequence type, OST）。通过MSP dendrogram聚类分析、MALDI Biotyper建立数据库、ClinPro Tools建立模型3种方法研究ST11分型的可能性。结果 MSP dendrogram聚类分析在距离水平线1000处可将80株菌聚类分析为两类,敏感性为65．78％,特异性为92．85％。 MALDI Biotyper数据库验证结果：18株ST11有1株菌分为OST,32株OST有2株菌被分为ST11,敏感性为89．47％,特异性为96．77％。 ClinPro Tools用其余菌株验证模型全部正确,敏感性和特异性均为100％。结论 MALDI?TOF MS可通过3种方法区别ST11与OST,以ClinPro Tools软件建立模型的方法最可靠。     

重组人酸性成纤维细胞生长因子C-末端测序

目的：应用蛋白质N-末端序列分析仪测定重组人酸性成纤维细胞生长因子（aFGF）C-末端氨基酸序列。方法：应用多肽分析软件选择合适的蛋白内切酶完全酶切aFGF，酶切后用RP—HPLC进行分离，收集软件预测C-末端肽段保留时间处的肽段峰，用蛋白质N-末端序列分析仪直接测得该肽段氨基酸全序列。结果：实测肽图图谱与软件预测理论图谱一致，所得到的肽段为aFGF C-末端肽段，经测序其结果与理论序列完全一致。结论：通过选择合适的蛋白内切酶，可运用RP—HPLC和蛋白质N-末端序列分析仪测定基因工程产品C-末端氨基酸序列。     

粒-巨噬细胞集落刺激因子治疗老年人抗肿瘤治疗所致粒细胞减少的疗效观察

重组人粒－巨噬细胞集落刺激因子（rhGM-CSF，生白能）是一种调节粒一单／巨噬细胞生长和功能的具有多种潜能的造血生长因子~[1、2]。80年代以来，临床应用证实rhGM－CSF不仅能刺激造血干细胞分化和增殖，而且有增强单核、巨噬细胞抗感染和抗肿瘤的功能。~[3、4]。本文就我科1995年7月至1997年3月应用rhGM－CSF（美国失灵葆雅公司生产）治疗20例老年恶性肿瘤病人因放疗或化疗所致粒细胞减少或缺乏56例次的疗效进行分析，探讨其临床价值。1资料与方法1．1一般资料：20例恶性肿瘤病人，均经手术或活检病理证实，其中男性14例，女性6例；…     

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。     

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。     

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.     

Isolation and chemical characterization of a toxin isolated from the venom of the sea snake, Hydrophis torquatus aagardi

Sea snakes (family: Hydrophiidae) are serpents found in the coastal areas of the Indian and Pacific Oceans. There are two subfamilies in Hydrophiidae: Hydrophiinae and Laticaudinae. A toxin, aagardi toxin, was isolated from the venom of the Hydrophiinae snake, Hydrophis torquatus aagardi and its chemical properties such as molecular weight, isoelectric point, importance of disulfide bonds, lack of enzymatic activity and amino acid sequence were determined. The amino acid sequence indicated a close relationship to the primary structure of other Hydrophiinae toxins and a significant difference from Laticaudinae toxins, confirming that primary toxin structure is closely related to sea snake phylogenecity.     

TsTX-IV, a short chain four-disulfide-bridged neurotoxin from Tityus serrulatus venom which acts on Ca2+-activated K+ channels.

The primary structure of TsTX-IV, a neurotoxin isolated from Tityrus serrulatus scorpion venom, is reported. Its amino acid sequence was determined by automated Edman sequential degradation of the reduced and carboxymethylated toxin and of relevant peptides obtained by digestion with Staphylococcus aureus strain V8 protease or trypsin and cleavage by CNBr. The complete sequence showed 41 amino acid residues, which account for an estimated molecular weight of 4520, and eight half-cystine residues which cross-link the toxin molecule with four disulfide bonds. The molecular weight determined by mass spectrometry was 4518. Comparison of this sequence with those from other scorpion toxins showed a resemblance with toxins which act on different types of K+ channels. TsTx-IV was able to block Ca2+-activated K+ channels of high conductance. TsTX-IV is the first four-disulfide-bridged short toxin from T. serrulatus so far completely sequenced.     

Structure verification of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody

Structure of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody was verified by the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS)and N-terminal sequencer. Molecular masses, N-terminal sequences and peptide maps of the antibody treated with different reagents and enzymes were measured. Results indicate that the amino acid sequences of light and heavy chains and 10 disulfide bonds were consistent with theoretical structure. By comparison of molecular masses and peptide maps for the fully glycosylated and deglycosylated samples, the N-linked glycosylation site was identified. The method is simple, rapid, precise, and could be referred to the quality control and structure determination of other IgG1 products.     

Characterisation of a secreted form of recombinant derived human growth hormone, expressed in Escherichia coli cells

Recombinant DNA derived human growth hormone (rhGH), Genotropin, has been expressed in E. coli cells as a pre-hormone, where the heat stable enterotoxin II signal peptide (STII) was linked to hGH to get secretion of the hormone to the periplasmatic space. The pre-hormone was efficiently cleaved during secretion, by an endogenous signal peptidase generating the correct N-terminal (Phe) end as shown by protein sequence analysis. The purity of rhGH was studied by SDS-PAGE, in combination with laser densitometry and HI-HPLC. These techniques showed that the level of modified rhGH forms, e.g. aggregated and proteolytically cleaved (16 and 6 kDa) in the preparation was in the 0.5-1% range. Furthermore, evidence that the correct disulphide bonds (Cys53-Cys165; Cys182-Cys189) were formed in rhGH during secretion has been shown by a combination of tryptic fingerprint and amino acid analysis. CD-spectroscopic analysis suggested an identical secondary structure to that of pituitary derived human growth hormone (pit-hGH). Isoelectric focusing revealed an isoelectric point (pI) for rhGH of 5.0 similar to pit-hGH and in excellent agreement with the theoretical value 5.1, based on the primary sequence. Finally, an apparent molecular weight of 22,000 was obtained for rhGH, by SDS-PAGE. All these physico-chemical studies suggest that rhGH is structurally identical to pit-hGH, somatotropin.     

Characterization of Heat-Labile toxin-subunit B from Escherichia coli by liquid chromatography–electrospray ionization-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The possibilities of characterizing the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) by liquid chromatography electrospray mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption with time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated. The B subunit from recombinant E. coli (expression in Pichia pastoris) can be detected by LC/ESI-MS expressed in P. pastoris and the charge envelope signals can be observed; LC/ESI-MS and MALDI-TOF-MS analysis allowed the acquisition of labile toxin subunit B (LTB) molecular weight and preliminary structural characterization of LTB toxin. MALDI-TOF analysis after reduction and alkylation of the protein evidenced the presence of one disulfide bond in the structure of the protein. Confirmatory analysis was carried out by detection of most of the tryptic fragments of the B subunit by MALDI-TOF-MS, obtaining total coverage of the protein sequence. Possible biovariations in the toxin can mostly be determined by sequencing, where an increase of molecular mass in the N-terminal side of the protein was identified. This modification may be due to an O-GlcNAc-1-phosphorylation.     

Primary structure,spectroscopic and inhibitory properties of a two-chain trypsin inhibitor from the seeds of charlock (Sinapis arvensis L), a member of the napin protein family

A protein with inhibitory activity toward trypsin has been isolated from Sinapis arvensis L (charlock). It has a molecular weight of 15500 and consists of two chains connected by disulfide bonds. The amino acid sequence was determined and showed that it belongs to the napin family of storage proteins. CD studies showed an α-helix content of 12% and a β-structure of about 50%.     

液相色谱-串联质谱法研究重组水蛭素12肽与瑞替普酶融合蛋白(HV12p-rPA)的一级结构

利用液质联用研究重组水蛭素12肽与瑞替普酶融合蛋白(HV12p-rPA)的一级结构。采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)测定水蛭素12肽与瑞替普酶融合蛋白(HV12p-rPA)的相对分子质量；采用液质联用分别分析HV12p-rPA的胰蛋白酶(trypsin)和胰凝乳蛋白酶(chymotrypsin)酶解产物。MALDI-TOF-MS测得HV12p-rPA的相对分子质量为41 472 Da，与理论值相符；HV12p-rPA的胰蛋白酶(trypsin)酶解产物进行液质联用分析结果表明该融合蛋白中含有瑞替普酶序列；HV12p-rPA的胰凝乳蛋白酶(chymotrypsin)酶解产物进行液质联用分析，结果表明融合蛋白中含有水蛭素12肽，并检测出融合蛋白中的链接肽(DEGGGSY)，融合蛋白的N末端MASDF和C末端LDWIRDNMRP；采用液质联用进行肽图分析，识别出的多肽匹配度Xcorr均超过1.5，部分多肽的Xcorr超过3.0，表明本实验测定过程中多肽识别结果准确可靠；目标蛋白的氨基酸序列覆盖率超过85%。结果确定了HV12p-rPA融合蛋白的序列与理论值相符。     

舟山眼镜蛇毒蕈碱样多肽MP的部分理化性质测定

目的对舟山眼镜蛇毒蕈碱样多肽MP成分进行纯化及部分理化性质测定。方法采用色谱技术纯化蛇毒MP成分,质谱仪测定其相对分子质量(Mr);Edman降解法分析部分氨基酸序列,与已知成分比对;以卵磷脂为底物测定MP组分的磷脂酶A2活性;采用Bliss法测定MP对小鼠的急性毒性。结果 MP的Mr为13 260,其N端16位氨基酸序列为NLYQFKNMIQCTVPSR,与已知蛇毒磷脂酶A2基本一致,并具有较强的磷脂酶A2活性;腹腔注射MP对小鼠的LD50值为14.3 mg/kg。结论 MP为一种眼镜蛇毒磷脂酶A2。