高效液相色谱/质谱法识别不同明胶酶解产物中特征多肽

在不同动物胶原蛋白序列比对基础上,利用高效液相色谱/质谱联用技术(HPLC/MS)建立一种通过特征多肽进行明胶鉴别的方法。实验以牛明胶和猪明胶为对象,序列比对结果表明,牛和猪的Ⅰ型胶原蛋白α1链和α2链均存在氨基酸序列差异。利用胰蛋白酶将牛明胶和猪明胶降解,通过HPLC/MS分析了两种明胶酶解产物中的多肽片段。结果表明:牛明胶和猪明胶酶解产物中均可检测出存在序列差异的特征性多肽,其氨基酸序列差异与两种胶原蛋白序列比对结果中的序列差异一致;酶解产物中特征多肽上的脯氨酸存在不同程度的羟基化修饰,特征多肽的相对含量主要受明胶分子量范围影响。利用HPLC/MS检测明胶酶解产物中的特征多肽是一种鉴别牛明胶和猪明胶的有效方法。     

纳升电喷雾串联质谱法确证阿托西班的一级结构

利用电喷雾串联质谱对合成八肽阿托西班进行了二硫键还原前后的精确分子量测定和一级结构的确证.首先通过全扫描模式测定了其还原前后的精确分子量,然后选择母离子m/z 498.73(双电荷)通过串联质谱(MS/MS)得到碎片离子,采用Y离子的方法测定了阿托西班的序列并对其的修饰位点进行了确证.本方法具有灵敏度高、速度快、样品无需纯化等特点,在多肽类药物一级结构分析方面具有独特的优势.     

纳升电喷雾串联质谱在阿托西班一级结构确证中的应用

利用电喷雾串联质谱对合成八肽阿托西班进行了二硫键还原前后的精确相对分子质量测定和一级结构的确证.首先通过全扫描模式测定了其还原前后的精确相对分子质量,然后通过串联质谱(MS/MS)得到母离子m/z 498.73(双电荷)的碎片离子,采用y离子的方法测定了阿托西班的序列并对其修饰位点进行了确证.该方法具有灵敏度高、速度快、样品无需纯化等特点,在多肽类药物一级结构分析方面具有独特的优势.     

反相高效液相色谱与质谱联用分析合成七肽的消旋产物

采用反相高效液相色谱(RP-HPLC)与质谱(MS)联用技术对固相法化学合成七肽(H2N-PFNSLAI-COOH,M r 760.9)时出现的消旋产物进行了分析。根据弱疏水性合成七肽粗品特性,提出了充分利用吸附和分配双重保留机制的“早期吸附”概念,以此优化色谱条件得到4个峰形良好的色谱峰;质谱分析表明:4谱峰具有相同的m/z761.5[M H] 值,对每一消旋产物进行在线多肽测序,并利用串联质谱(MSn)谱图叠加的方法,得到b轴和y轴两套片段信息,成功确定了合成七肽的4个消旋产物。     

含有28个氨基酸的复杂多肽的串级质谱全序列分析研究

利用MALDI-TOF/TOF MS和ESI-MS/MS对一种含有多达28个氨基酸的复杂合成多肽成功进行了全序列测定. 通过调节激光强度、碰撞诱导解离(CID)能量等质谱参数以及依据不同序列分析软件, 获得了涵盖所有b型和y型碎片离子的串级质谱图. 结果显示这种方法可以有效地解决de novo测序方法遇到的谱峰过于复杂导致运算死机等问题. 通过讨论如何对含有超过20个氨基酸片断的多肽进行合格串级质谱实验, 为蛋白质组学肽段序列测定提供了新的方法和思路.     

合成多肽的电喷雾质谱研究

利用电喷雾多极串联质谱对3种合成多肽进行了系统的鉴定和分析研究。首先通过全扫描模式测定了其分子量，然后选择[M H]^ 或[M 2H]^2 离子通过串联质谱(MS／MS)得到碎片离子，采用y离子和b离子互补的方法测定了多肽序列。利用文献数据对这种方法进行了验证，实验结果表明，该方法简便、快速、实用。     

基于生物质谱技术的磷酰化修饰策略在多肽测序中的应用

该文建立了一种利用磷酰化修饰结合电喷雾质谱(ESI-Q-TOF)测定多肽氨基酸序列的有效方法。利用Atherton-Todd反应,以二丙基亚磷酰酯(DPP)为磷酰化试剂,应用生物质谱技术,对磷酰化修饰后的5种模型肽的磷酰化反应情况进行了系统研究,考察了磷酰化肽的二级质谱特征,并与未经磷酰化反应的肽的二级质谱特征对比。结果表明,经过磷酰化修饰后,肽的二级质谱中的a1离子信号强度明显增加,可以准确鉴定其N端氨基酸;b系列离子信息完整,信号强度增强,使得多肽C ID测序的谱图简单、清晰,有利于肽的氨基酸序列的测定;赖氨酸(K,128.10 u)和谷氨酰胺(Q,128.13 u)两种氨基酸质荷比相近,由于二者磷酰化修饰后的差异性,使其得到准确区分。经过5种已知氨基酸序列的模型肽的磷酰化后结合质谱技术进行氨基酸序列测定验证,结果表明该方法简单、快速、准确,提高了利用质谱技术进行多肽测序的准确度和灵敏度,可为蛋白质组学研究提供有效的技术手段。     

牡蛎源类蛋白反应修饰肽的分离纯化及肽锌螯合物的结构表征

以牡蛎为原料制备了类蛋白反应修饰肽,利用Sephadex G-15凝胶层析柱和反向高效液相色谱(RP-HPLC)等分离技术得到1条锌离子螯合活性为161 mg/g的多肽(M_w=835),多肽序列为EVPPEEH.以测得的肽序列为模板合成多肽,将纯肽与锌离子进行螯合反应制备肽锌螯合物.螯合物的红外光谱和圆二色光谱表征结果表明,锌离子主要与多肽链上的羰基氧发生相互作用.与多肽的空间结构相比,螯合物的无规则卷曲结构减少,β转角增加而β折叠减少.由肽锌螯合物的分子模拟和二级质谱结果可知,多肽与锌离子螯合后有2种空间构象:一种通过六配位的方式螯合1个锌离子,其中主要的螯合位点为多肽Val-2和Pro-3或者Glu-5和Glu-6之间的羰基氧;另一种是通过四配位的方式螯合1个锌离子,主要的螯合位点为多肽Glu-5和Glu-6之间的羰基氧.     

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。     

基质辅助激光解析电离质谱和电喷雾质谱共同快速确证达托霉素结构

运用基质辅助激光解析电离-飞行时间串联质谱(MALDI-TOF/TOF MS)和电喷雾-四级杆-飞行时间质谱(ESI-Q-TOF MS)快速确证环脂肽达托霉素的结构。首先,ESI检测达托霉素相对分子量为1619.7107,与理论值偏差0.0007。选择其双电荷峰m/z 809.848作为母离子进行ESI串联质谱(MS/MS)测定,成功匹配了达托霉素环外氨基酸序列C9H19CO-Trp-Asn-Asp。其次,优化Li OH裂解达托霉素的实验条件,以MALDITOF/TOF MS监测开环效果,获得95%以上的开环样本后,分别运用MALDI和ESI进行MS/MS测定,达托霉素开环产物的b+和y+全部被匹配,达托霉素全部氨基酸序列得到确证。最后,对开环产物的ESI-MS/MS条件进一步优化,获得了丰富的低端碎片离子,解析了脂肪酸链结构,并绘制了脂肪酸链的裂解图。本方法快速、简便、准确,是确证环脂肽类化合物结构的可靠方法。     

Detection and characterization by high-performance liquid chromatography and mass spectrometry of two truncated goat alphas2-caseins

The identification and characterization of truncated forms of goat alphas2-Cn variants A and E are reported. The two proteins, which have experimental Mr values of 24 183 and 24 227 Da, were detected as minor components in a goat milk sample from an autochthonous breed of southern Italy, 'Rossa Mediterranea', by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). Characterization of the amino acid sequences, performed by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), RP-HPLC/ESI-MS and tandem mass spectrometry (MS/MS), demonstrated that the polypeptide chains correspond to the 1-204 sequence of mature alphas2-Cn variant A (component with Mr of 24 183 Da) and E (component with Mr of 24 227 Da), respectively. These components seem to be the product of a differential splicing of pre-messenger RNA during the translation process of the alphas2-Cn variants A and E.     

反相高效液相色谱/电喷雾质谱法分析化学合成七肽粗产物

利用RP—HPLC／ESI—MS直接分析用芴甲氧羰基(Fmoc)固相多肽合成方法在PHB树脂上偶联合成的一个七肽(H2N—Tyr—Val—Asn-Thr-Asn—Met—Gly—COOH,Mr797．3)粗产物。RP—HPLC显示合成粗产物含有1个主成分,4个次要成分和多个微量成分;与之联用的电喷雾质谱则同步准确地测定出各成分的分子量(m／z)并自动对各主要成分的化学结构进行了串联质谱分析。结果证明,粗产物中的主成分即为目标七肽,另外几个主要副产物为七肽的氧化产物或残缺肽。     

Identification and characterization of a new beta-casein variant in goat milk by high-performance liquid chromatography with electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry

A new variant of beta-casein was detected in the casein fraction obtained from milk of a goat belonging to an autochthonous breed of southern Italy, "Argentata dell'Etna". Reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) analysis indicated that the new beta-casein variant, here named D, has a M(r) 15 Da higher than that of variant C previously described. The modification in the amino acid sequence responsible for the 15 Da difference in M(r) between variants C and D was determined by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and RP-HPLC/ESI-MS, and it was demonstrated that it is due to the point mutation Val(207) --> Asn(207). The phosphorylation pattern of the new variant D was shown to be identical to that of variant C, as the protein shows two phosphorylation levels, 5 and 6P, occurring with comparable relative abundances. Ser35 was determined as one of the phosphorylation sites, whereas the others were probably analogous to those determined previously for the beta-Cn variant C, at Thr12 and Ser1517-19. The results reported here indicate that the combined use of RP-HPLC/ESI-MS, MALDI-TOFMS and MS/MS represents a powerful tool for the detection and characterization of minor components present in complex protein mixtures.     

Identification of allergenic milk proteins markers in fined white wines by capillary liquid chromatography–electrospray ionization-tandem mass spectrometry

A method based on capillary liquid chromatography combined with electrospray ionization-tandem mass spectrometry (CapLC–ESI-MS–MS) for the detection and identification of casein deriving peptides in fined white wine is described. This is the first step towards the development of a liquid chromatography mass spectrometric method for the detection/identification of markers of potentially allergenic milk proteins used as wine fining agents. The method demonstrated to be capable of detecting some peptides arising from α and β casein (with the relative aminoacidic sequences elucidated) in extracts of white wine fined with casein at 100 and 1000 μg/mL. This MS based approach appears to be a useful tool for screening purposes as well as a confirmatory tool for the unequivocal identification of caseins in ELISA positive samples.     

Quantification of bovine casein fractions by direct chromatographic analysis of milk. Approaching the application to a real production context

The ability to quantify the casein content by an exact and cost-effective approach represents an issue of crucial importance in the dairy industry as the natural variations in milk protein concentration can markedly affect the yield of the cheesemaking processes, thus causing a direct and significant economic impact on the producers. In this work, the separation and quantification of α_s1-, α_s2-, κ- and β-casein was carried out by direct RP-HPLC analysis of milk. The identification of each casein was established by electrospray ionization mass spectrometry. The data show that this method is able to effectively separate the bovine casein fractions, it provides simplified analytical conditions (with special regard to mobile phase composition and gradient profile) and faster separation while ensuring adequate precision to achieve reliable quantifications in milk samples from dairy production.     

应用双向电泳和质谱联用技术研究乳源蛋白的差异性

应用双向电泳和质谱联用技术,对不同乳源蛋白的差异性进行了研究。根据ImageMaster 2DPlatinum图像分析软件对不同乳源酪蛋白和乳清蛋白的双向电泳(2-DE)图谱进行蛋白斑点的匹配分析,获得21个存在于水牛奶中主要分布在低丰度蛋白区的酪蛋白差异蛋白点和24个存在于水牛奶中乳清蛋白差异蛋白点。这些差异蛋白点经质谱鉴定分析,得到4个属于水牛奶酪蛋白的主要组分和2个与水牛奶中酪蛋白有较高同源性的新组分,同时获得4个属于水牛奶乳清蛋白的主要组分和3个与水牛奶中乳清蛋白有较高同源性的组分。     

Direct analysis of oligosaccharides and alpha hydroxy acids in fruits using electrosonic spray ionization mass spectrometry

Electrosonic spray ionization (ESSI) is a derivative technique of electrospray ionization (ESI) for mass spectrometry (MS) in which droplets are charged in the course of sonic spray. In this study, we applied ESSI MS to direct analysis of oligosaccharides and alpha hydroxy acids (AHAs) in fruits. The components were extracted from fruit fleshes by a feasible method prior to ESSI MS analysis, but the fruit juices were analyzed without further pretreatment. The results demonstrate that mainly alkali metal adducts of oligosaccharides are favorably produced in positive ion mode, while deprotonated AHAs and oligosaccharides are produced in negative ion mode. Compared with mass spectra obtained using electrospray droplet impact/secondary ion mass spectrometry (EDI/SIMS), mass spectra using ESSI make the identification of oligosaccharides more straightforward in positive ion mode than in negative ion mode.     

Detection and characterization by high-performance liquid chromatography and mass spectrometry of a goat beta-casein associated with a CSN2 null allele

The identification and characterization of a truncated goat beta-casein, associated with a null beta-casein allele (CSN2(O')), is reported. The truncated beta-casein predicted at the DNA level (NCBI Acc. No. CAB39313) but never observed at the protein level, here named beta-casein O, was detected as a minor component in a goat milk sample from an autochthonous breed from southern Italy, 'Rossa Mediterranea', by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). The ESI mass spectrum of the intact beta-casein O determined an M(r) value of 18 780 Da (calculated 18 781.5). Characterization of the amino acid sequence, performed by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), RP-HPLC/ESI-MS and tandem mass spectrometry (MS/MS), demonstrated that the amino acid sequence corresponds to the 1-166 sequence of mature beta-casein variant A (Acc. No. P33048), thus confirming that the protein is coded by the null allele CSN2(O'), characterized by a transition (C --> T) at the 373rd nucleotide of the 7th exon of the gene, which generates a premature stop codon in position 182.     

Simultaneous analysis of thirteen diuretics residues in bovine milk by ultra-performance liquid chromatography/tandem mass spectrometry

A simple, rapid, sensitive and specific method used to screen and confirm multi-class diuretics residues in whole bovine milk is described. Thirteen drugs of four different classes including carbonic anhydrase inhibitors, loop, thiazide and potassium-sparing diuretics were extracted from whole milk by acetonitrile followed by further purification with hexane. The analytes were separated using an ACQUITY UPLC BEH C18 column and detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS). MS data acquisition was performed by a time-scheduled multiple reaction monitoring program, selecting two ion transitions for each target compound. The overall average recoveries based on matrix-fortified curves fortified with diuretics at three levels ranged from 80.6 to 108.8% with the coefficients of variation ranging from 2.6 to 19.7% (n = 6). The limits of quantitation (LOQs) of diuretics in bovine milk were 5.0 microg/kg for spironolactone and 0.5 microg/kg for other analytes, respectively.