基质辅助激光解吸附电离-飞行时间串联质谱研究重组人促红细胞生成素一级结构

建立了生物质谱研究rhEPO一级结构的系列方法,包括分子量、唾液酸含量以及N-糖含量等糖基化性质的测定方法,并用于4种国产rhEPO样品的分析;在此基础上优化酶切条件,使测定的肽质量指纹谱(PMF)的覆盖率均达到98%,成功鉴定了4个样品中的全部N-糖基化位点以及对分子中两对二硫键进行了结构确证,测定4个样品分子量分别为27754.1±27.4Da、27941.4±23.2Da、27682.5±22.0Da和27914.7±22.5Da;唾液酸含量分别为5.0%、4.8%、5.6%和5.4%;糖含量分别为34.3%、34.7%、34.1%和34·6%。此外还对4种产品在O-连接糖型及N-连接糖型上的区别做了初步探讨。     

人肝癌Bel-7402细胞及Bel-7402/5-FU细胞的N-连接聚糖的差异分析

以培养的人肝癌细胞Bel-7402及其5-氟尿嘧啶(5-FU)耐药细胞(Bel-7402/5-FU)为研究对象,通过使用快速PNGase F酶切,结合TMPP-Ac-OSu和甲胺化共衍生方法,对两者的总蛋白和分泌蛋白的N-连接聚糖进行了基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)分析。从两种细胞总蛋白中共鉴定到56种N-连接聚糖,分泌蛋白中鉴定到38种N-连接聚糖。5-FU与Bel-7402相比,耐药细胞岩藻糖化唾液酸糖型在总蛋白和分泌蛋白中都显著升高;高甘露糖型N-聚糖在总蛋白中下降,而在分泌蛋白中显著增加。本研究为进一步探索肿瘤耐药提前诊断提供糖链标志物起到一定的参考作用,并为后续解决癌症治疗的耐药提供理论依据。     

基于电喷雾电离质谱的人肝癌细胞HepG2与正常肝细胞L02的N-糖链的定性定量比较

以培养的原发性肝癌细胞HepG2和正常肝细胞L02为研究对象,采用细胞裂解液提取总蛋白,用PNGase F酶解释放N-糖链,以微晶纤维素柱结合石墨碳柱纯化分离N-糖链,通过电喷雾电离质谱(ESI-MS)和串联质谱(MS/MS)对N-糖链进行序列鉴定,以β-环糊精为内标对2种细胞系的N-糖链进行了定量比较分析.结果表明,在肝癌细胞系HepG2和正常细胞系L02中共检测到26种N-糖链,与L02相比,HepG2的大多数高甘露糖型糖链、唾液酸化糖链和岩藻糖基化糖链的数量都明显升高,其中有15种糖链在数量上具有极显著性差异(p0.01),1种糖链具有显著性差异(p0.05).本研究为进一步探索肝癌中各类N-糖链的表达特点及发现早期肝癌糖链标志物提供了参考.     

IgG1型单克隆抗体对照品的一级结构解析

对单克隆抗体药物对照品(IgG1型)进行了全面的结构表征。采用Exactive plus EMR质谱测定了去N糖前后抗体的精确分子量,并通过计算N糖含量得出其完整分子量为148 285.0～149 020.8,完整去糖后分子量为145 810.4,N糖含量为1.67%～2.15%。优化了全柱成像实时等电聚焦毛细管电泳(WCID-cIEF)检测方法,结果显示该抗体等电点分布在8.21～8.74之间,其中主成分等电点为8.61,相对含量为61.43%。继而使用离子交换色谱检测了电荷异质性,发现碱性峰含量为4.1%,酸性峰含量为23.9%,主峰含量为72.0%,与WCID-cIEF结果吻合,并证明了方法间良好的重现性。通过切糖前后的肽图分析,获得糖肽分布信息,识别出糖修饰位点及重轻链的互补决定区(CDR),甲硫氨酸(M)的氧化位点及氧化率,并通过抗体还原前后的肽图解析出二硫键连接。研究结果同时提示该抗体对照品无C末端糖化修饰现象。     

气相色谱-质谱法测定鸡肉中残留的环丙胺嗪及其代谢物三聚氰胺

建立了鸡肉中残留的环丙胺嗪及其代谢物三聚氰胺的气相色谱-质谱(GC-MS)检测方法。样品经酸化的乙腈-水溶液提取、二氯甲烷去脂、混合型的阳离子交换(MCX)固相萃取柱净化、N,O-双三甲基硅基-三氟乙酰胺(BSTFA)衍生化后进行GC-MS测定。环丙胺嗪采用外标法定量,三聚氰胺以三聚氰胺-15N3为内标定量。结果表明,环丙胺嗪与三聚氰胺的线性范围均为100～1000 μg/L,定量限为20 μg/kg。在20,40,80 μg/kg 3个添加浓度下目标化合物的平均回收率为75%～110%,批内、批间相对标准偏差分别小于10%和15%。该方法样品前处理简单,测定灵敏度高,定性准确,选择性好,可实现鸡肉中环丙胺嗪与三聚氰胺残留的同时测定。     

Determination of mannose in honey by isotope dilution gas chromatography mass spectrometryEI北大核心CSCD

建立了一种同位素稀释-气相色谱/质谱(GC-MS)测定蜂蜜中甘露糖含量的方法。选择了一种新型化合物[13C;D]-D-甘露糖作为蜂蜜中甘露糖检测的同位素内标,优化了甘露糖及其内标的气相色谱分离条件和质谱参数。蜂蜜样品经70%乙醇水溶液溶解后加入同位素内标,采用两步衍生法对蜂蜜中糖组分进行衍生化,即先加入2.5%盐酸羟胺-吡啶溶液进行肟化衍生,再加入N,O-双(三甲基硅基)三氟乙酰胺(BSTFA)进行硅烷化衍生,采用GC-MS对甘露糖进行定性分析和定量检测。结果表明,以洋槐蜜、油菜蜜、荆条蜜和椴树蜜为代表性样品基质,甘露糖的检出限均为6 mg/kg,定量限为20 mg/kg,在20,40,200 mg/kg 3个添加水平下方法的平均回收率在66.1%~91.7%之间,相对标准偏差不超过20%。该方法适用于不同类型蜂蜜中甘露糖含量的测定。     

定量核磁共振波谱法测定3种低聚果糖的含量北大核心CSCD

以蔗糖为内标物,氘代水为溶剂,采用氢-1核磁共振波谱法(1H-NMR)测定蔗果七糖、蔗果八糖、蔗果九糖等3种低聚果糖(FOS)的含量。优化后的1H-NMR参数如下:温度25℃,谱宽(2~14)×10^(-6),脉冲角度45°,脉冲延迟时间5 s,采样时间3 s,扫描次数16次。对蔗果七糖、蔗果八糖和蔗果九糖样品进行了仪器精密度和重复性试验,测定值的相对标准偏差(n=6)均小于0.50%。方法用于测定3种样品含量,测定结果与质量平衡法所得结果进行了比较,经F检验法评估,两种方法具有显著性差异。但由于定量核磁共振波谱法作为绝对含量的测定方法,仅需常见的化学品即可直接对待测成分进行测定,可以佐证质量平衡法的赋值结果。     

基于~1H-NMR、~(31)P-NMR的三苯基膦三间磺酸钠定量分析研究

该文建立了三苯基膦三间磺酸钠(TPPTS)的核磁氢谱(~1H-NMR)、核磁磷谱(~(31)P-NMR)、定量分析方法。通过~1H-NMR、~(13)C-NMR、~(31)P-NMR并结合~1H-~1H COSY、~1H-~(13)C、~1H-~(13)C HMBC对TPPTS的氢原子、碳原子以及磷原子的化学位移进行归属;氢谱定量选用三■烷作内标、氘代N,N-二甲基甲酰胺(DMF-D_7)作溶剂,磷谱定量选用KH_2PO_4作内标、氘代水(D_2O-D_2)作溶剂,通过对混合体系中各自旋核纵向弛豫时间(T1)的测定,为弛豫延迟时间(D1)的合理设置提供依据。以上两种方法对体系中TPPTS的定量分析结果分别为(58.72±0.21)%、(58.51±0.21)%,测定结果一致性高、平行性好,且检测过程无需待测组分的标准品,能实现TPPTS含量的快速准确测定。     

液相色谱-串联质谱法测定育肥猪配合饲料中的阿奇霉素

通过对提取溶剂、净化方法及色谱-质谱条件的优化,建立了配合饲料中阿奇霉素(AZM)的液相色谱-串联质谱测定方法。样品经乙腈超声波提取,MCX固相萃取柱净化,BDS Hypersil C_(18)(2.4μm,100 mm×2.1 mm)色谱柱分离,以甲醇-0.05 mol/L乙酸铵水溶液为流动相梯度洗脱,正离子模式电离,多反应监测模式检测,同位素内标法定量。在优化条件下,AZM在1~250μg/L范围内线性关系良好,相关系数(r~2)为0.999 1,检出限(S/N≥3)为2.8μg/kg,定量下限(S/N≥10)为8.4μg/kg;在0.5,1,5,10 mg/kg加标水平下,回收率为87.0%~106.6%,批内相对标准偏差为0.58%~5.4%,批间相对标准偏差为3.1%~5.4%。该方法准确、灵敏,选择性强,基质干扰小,可用于配合饲料中AZM的测定。     

固相萃取/高效液相色谱-串联质谱法测定水产品中氨苯砜及其代谢物残留量

建立了高效液相色谱－串联三重四极杆质谱（HPLC－MS/MS）测定水产品中氨苯砜及其代谢物残留量的方法。样品加入D8－氨苯砜内标,经1%氨化乙腈提取,正己烷去脂后,采用MCX阳离子固相萃取柱进行富集和净化,氮吹浓缩至0.2 mL左右。用甲醇和水作为流动相,以CAPCELL PAK C18色谱柱（2.0 mm × 100 mm,3 μm）进行分离,在质谱多反应监测（MRM）、正离子电离模式下测定,采用内标法定量。氨苯砜和N-乙酰氨苯砜在0.1 ~ 5.0 μg/kg范围内线性关系良好,相关系数（r2）均大于0.99,方法检出限（LOD）为0.1 μg/kg,定量下限（LOQ）为0.2 μg/kg。以不同类型的水产品为空白基质,在0.2、1.0、2.0 μg/kg加标水平下,平均加标回收率为94.0% ~ 109%,相对标准偏差（RSD）为2.7% ~ 11%。用该法对药代实验中获得的阳性肌肉样品进行测定,通过对化合物的保留时间和离子对信息进行鉴别,鉴定出氨苯砜的代谢产物为N-乙酰氨苯砜。该方法的检出限低、精密度好、准确度高,能够实现对不同水产品中氨苯砜残留量的准确定性与定量检测。     

Characterization of N- and O-glycopeptides of recombinant human erythropoietins as potential biomarkers for doping analysis by means of microscale sample purification combined with MALDI-TOF and quadrupole IT/RTOF mass spectrometry

The structural characterization of the O- and N-glycan structures of three different commercially available recombinant human erythropoietins (rhEPOs) is represented by means of a microscale sample purification using ZipTip technology and MALDI-TOF and MALDI low-energy CID MS. Glycopeptides were released from rhEPO samples by a differential endoproteolytic digestion to obtain site-specific glycosylation patterns. Mass accuracies in the range of +/- 0.04% obtained by the high-resolution TOF instrument allowed an unambiguous assignment of N-glycan structures via glycan database software. Furthermore, the O-glycan structures were directly analyzed on the glycopeptide level by MS/MS experiments. Principally, site-specific glycosylation was found to be very similar for the three different rhEPOs (EPO-alpha, EPO-beta, and novel erythropoiesis stimulating protein (NESP)) but exhibiting quantitative differences in distinct O- and N-glycan moieties. Significant differences were found in the degree of sialylation and acetylation. Especially, a considerable degree of variation of the O-acetylation of sialic acid residues could be realized on the glycan structures of O- and N-glycopeptides, whereas EPO-alpha and EPO-beta could be clearly differentiated from NESP solely on the O-glycopeptide level.     

通过式固相萃取净化/液相色谱-串联质谱法快速检测水产品中11种青霉素残留

采用通过式固相萃取净化策略去除样品基质中的脂肪和磷脂等杂质干扰,结合液相色谱-串联质谱检测,建立了水产品中11种青霉素残留的同时快速分析方法。样品经80%乙腈水溶液提取,Oasis PRi ME HLB通过式固相萃取柱净化,C_(18)色谱柱分离,0.05%甲酸乙腈溶液和0.05%甲酸水溶液梯度洗脱,多反应监测正离子模式扫描,内标法定量。11种目标物在相应浓度范围内线性关系良好,相关系数不低于0.99,检出限为0.30~1.5μg/kg。基质加标回收率为85.5%~110%,相对标准偏差(RSD)为5.9%~14.3%。该方法前处理操作简便,灵敏度和准确度高,可实现水产品中多种青霉素药物残留的同时快速测定。     

超高效液相色谱-串联质谱法测定蔬菜和水果中腈吡螨酯残留量

建立了蔬菜和水果中腈吡螨酯残留量的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法。样品经乙腈均质提取,混合使用乙二胺-N-丙基硅烷(PSA)和C18两种基质分散净化剂净化,净化液过膜后直接进行UPLC-MS/MS检测。通过考察腈吡螨酯在不同果蔬样品中的基质效应发现,采用UPLC-MS/MS检测蔬菜和水果中腈吡螨酯残留量时,腈吡螨酯在不同种类样品中存在不同程度的基质减弱效应,采用空白基质溶液稀释标准建立校正的标准曲线外标法定量以消除基质效应。结果表明,腈吡螨酯在0.05~10μg/L浓度范围内具有良好的线性关系,相关系数不低于0.999 6。在0.000 05~2 mg/kg范围内进行加标回收率实验,平均回收率为81.1%~99.3%,相对标准偏差为4.7%~9.3%,方法的定量下限为0.043μg/kg,检出限为0.013μg/kg。     

QuEChERS/超高效液相色谱-四极杆-飞行时间质谱法同时测定小麦粉中17种农药残留

建立了超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF/MS)同时测定小麦粉中17种农药残留的检测方法。样品采用QuEChERS方法进行前处理,以0.1%(体积分数)乙酸乙腈溶液提取,PSA和C18吸附剂净化,电喷雾正离子模式检测,外标法定量,同时建立了包含一级精确质量和二级碎片离子信息的筛查确证谱库。在各自的线性范围内,17种化合物的线性关系良好,相关系数均大于0.995。方法的定量下限为5.0~10.0μg/kg,3个加标水平下的回收率为70.3%~114.9%,相对标准偏差(RSD,n=6)为0.4%~11.7%。该方法操作简便、稳定性好,具有较强的实际应用价值。     

MALDI-MS analysis of sialylated N-glycan linkage isomers using solid-phase two step derivatization method

Sialic acids usually locate at the terminal of many glycan structures in either α(2,3) or α(2,6) linkage, playing different roles in various biological and pathological processes. Several linkage specific carboxyl derivatization methods have been reported to discriminate between α(2,3) and α(2,6)-linked sialic acids by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Among them, ethyl esterification was recently reported to achieve linkage specific derivatization between α(2,3) and α(2,6)-linked sialic acids with good selectivity. However, the method suffered from the instability of the generated lactones and byproducts of the derivatives. To overcome these shortcomings, a solid-phase two step derivatization method was introduced to convert the α(2,6)-linked sialic acid into ethyl esters and the α(2,3)-inked counterparts into N-methyl amides, respectively. Under the optimized derivatization conditions, our method was able to achieve accurate relative quantification of N-glycan as well as their corresponding sialylated linkage types, superior to the ethyl esterification method. The solid phase derivatization strategy was further applied to investigate N-glycans from biosimilar antibody drug and human serum from patients and healthy volunteers. This method has the potential to be used in the biomarker discovery and pharmaceutical industry.     

固相萃取/高效液相色谱-串联质谱法同时检测环境水样中24种农药残留

建立了一种固相萃取/高效液相色谱-串联质谱(SPE/HPLC-MS/MS)同时检测水体中24种农药的分析方法。样品用乙腈提取后,经固相萃取小柱富集净化。以乙腈-0.1%(体积分数)甲酸水溶液为流动相梯度洗脱,在电喷雾离子源正离子模式下(ESI+)采用多反应监测(MRM)模式检测。结果显示,24种农药在1~200μg/L范围内具有良好的线性关系,相关系数(r2)均不小于0.998,水样中3个添加水平(5、20、100μg/L)下的回收率为65.9%~127.8%,相对标准偏差(RSD)为0.7%~14.2%;方法检出限为0.05~0.71 ng/L。采用该方法对大连地区10个河流入海口及2个水库的水样进行了检测,12个站位的样品中共检出10种农药,质量浓度为0.2~558.3 ng/L。结果表明,所建立的SPE/HPLC-MS/MS方法高效、灵敏、可靠,可用于实际水体中多种农药的同时检测。     

高效液相色谱-四极杆/离子阱质谱确证测定面粉及面制品中氨基脲

采用高效液相色谱-四极杆/离子阱质谱(HPLC-QTRAP-MS)建立了面粉及面制品中氨基脲的确证及测定方法。样品采用盐酸(HCl)提取,在超声辅助下与衍生剂邻硝基苯甲醛反应。衍生产物在中性条件下经PLS固相萃取柱净化、乙酸乙酯洗脱,经Shim-Pack XR-ODSⅢC18柱(2.0 mm×50 mm,1.6μm)分离,0.1%(体积比)甲酸-水溶液和甲醇溶液为流动相梯度洗脱;采用多反应监测(MRM)-信息依赖性采集(IDA)-增强子离子扫描(EPI)模式检测,EPI谱库确认,内标法定量。结果表明:氨基脲在0.5~40μg/L范围内线性关系良好(r=0.996);检出限(LOD,S/N=3)为0.10μg/kg,定量下限(LOQ,S/N=10)为0.25μg/kg;4个加标水平(0.25,0.5,2.0,10.0μg/kg)下的回收率为89.1%~112.8%;相对标准偏差(RSD)为1.4%~8.6%。该方法分析速度快,灵敏度高,回收率好,可用于面粉及面制品中氨基脲的快速检测。     

Glycoproteomic analysis and molecular modeling of haptoglobin multimers

Extra-thiol groups on the α-subunit allow haptoglobin (Hp) to form a variety of native multimers which influence the biophysical and biological properties of Hp. In this work, we demonstrated how differences of multimeric conformation alter the glycosylation of Hp. The isoform distributions of different multimers were examined by an alternative approach, i.e. 3-D-(Native/IEF/SDS)-PAGE, which revealed differences in N-glycosylation among individual multimers of the same Hp sample. Glycomic mapping of permethylated N-glycan indicated that the assembled monomer and multimeric conformation modulate the degree of glycosylation, especially the reduction in terminal sialic acid residues on the bi-antennary glycan. Loss of the terminal sialic acid in the higher order multimers increases the number of terminal galactose residues, which may contribute to conformation of Hp. A molecular model of the glycosylated Hp multimer was constructed, suggesting that the effect of steric hindrance on multimeric formation is critical for the enlargement of the glycan moieties on either side of the monomer. In addition, N241 of Hp was partially glycosylated, even though this site is unaffected by steric consideration. Thus, the present study provides evidence for the alteration of glycan structures on different multimeric conformations of Hp, improving our knowledge of conformation-dependent function of this glycoprotein.     

Fragmentation characteristics of permethylated oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight (TOF/TOF) tandem mass spectrometer

Matrix-assisted laser desorption/ionization two-stage time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS) was applied to characterize permethylated oligosaccharides. Under these ionization conditions such derivatives yield intense signals corresponding to sodium-cationized molecular species. A systematic study was conducted on a series of neutral and sialylated permethylated oligosaccharides to allow rationalization of the fragmentation processes. The major fragments observed in the MALDI-TOF/TOF-MS/MS spectra result from cleavage of glycosidic bonds, preferentially at N-acetylhexosamine and sialic acid residues. The fragments originating from both the reducing and the non-reducing ends of the glycan yield information on sequence and branching. Cross-ring cleavages, which are very informative of the linkages of the monosaccharide residues constituting these oligosaccharides, and 'internal' cleavage ions which are derived from elimination of substituents from around the pyranose ring, were also observed. This extensive fragmentation was shown to be useful for the structural characterization of oligosaccharides. MALDI-TOF/TOF-MS/MS of permethylated oligosaccharides appears to be a powerful tool for carbohydrate structural analysis.     

粮谷中11种二硝基苯胺类除草剂残留量的气相色谱-串联质谱法测定

建立了粮谷中11种二硝基苯胺类除草剂残留量的气相色谱-串联质潜(GC-MS/MS)测定方法,样品经乙腈提取、QuEChERS法净化,采用GC-MS/MS在多反应监测模式下进行快速分析,外标法定量.在优化实验条件下,11种二硝基苯胺类除草剂的线性范围均为1.0～20.0μg/L,相关系数大于0.996,方法定量下限为5μ...