Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

Preparation and Properties of Monoclonal Antibodies to Recombinant HBsAg Produced by Silkworm Larvae

Monoclonal antibodies to recombinant HBsAg produced by silkworm (Bombyx mori) larvae were prepared. The cross reactivity of the prepared antibodies was studied by solid-phase enzyme-linked immmunosorbent assay. It has been found that the prepared antibodies interact with recombinant and plasma HBsAg. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 474–476, September–October, 2005.     

Complete Mapping of a Cystine Knot and Nested Disulfides of Recombinant Human Arylsulfatase A by Multi-Enzyme Digestion and LC-MS Analysis Using CID and ETD

Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS³ (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138–Cys154, Cys143–Cys150, Cys282–Cys396, Cys470–Cys482, Cys471–Cys484, and Cys475–Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70 %). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.      

Quantitation of Indomethacin in Serum by HPLC Using Fluorescence Detection

Abstract

An improved, sensitive and accurate HPLC procedure using fluorescence detection for quantitation of indomethacin in serum has been developed. After addition of an equal volume of phosphate buffer, pH 6.6 to serum along with the internal standard, the samples were extracted with methylene chloride. Prior to chromatography, the extracted indomethacin was deacylated to its fluorescent product (DBI) in 0.01 N-NaOH. The mobile phase consisted of methanol and pH 4 acetate buffer (3:7 V/V) and the separation was achieved on a C18 reversed phase column. The retention times of DBI and the internal standard were 7.5 and 16.0 min. respectively. The fluorometric excitation and emission wavelengths were 278 and 358 nm, respectively. The sensitivity of the assay was 1 ng/ml of serum and the CV at this concentration was 2.46%. The standard plot was linear (r > 0.999) for indomethacin concentrations between 1 and 500 ng/ml. The inter-and intra-day studies showed high reproducibility (CV = 2.8%, F = 0.89, p > 0.05). The method was used to determine the complete serum level vs. time profiles of indomethacin in animals.     

Quantification of Monoclonal Antibodies from Bioreactor Supernatants Using Protein-G Sepharose Chromatography

A protein-G sepharose affinity chromatography was validated to quantify antibodies from cell culture supernatants. The calibration curve linear range was 30–480 μg immunoglobulin (IgG). Between-run study demonstrated a coefficient of variation 5.73–9.02% in 20 purification cycles and sample dilution with protein-free medium (>3 mL) showed a marked IgG amount over estimation. Sensitivity was 30 μg, and bovine serum albumin addition to columns did not affect IgG recovery (?90%). CB.Ifn-2.4 antibody quantification in MiniPERM^® bioreactor supernatant reached 1.5 mg mL^?1, coinciding with concentration measured by ELISA and thus demonstrating the protein-G sepharose column accuracy used to quantify IgG under these experimental conditions.     

Monoclonal Antibodies Application in Lateral Flow Immunochromatographic Assays for Drugs of Abuse Detection

Lateral flow assays (lateral flow immunoassays and nucleic acid lateral flow assays) have experienced a great boom in a wide variety of early diagnostic and screening applications. As opposed to conventional examinations (High Performance Liquid Chromatography, Polymerase Chain Reaction, Gas chromatography-Mass Spectrometry, etc.), they obtain the results of a sample’s analysis within a short period. In resource-limited areas, these tests must be simple, reliable, and inexpensive. In this review, we outline the production process of antibodies against drugs of abuse (such as heroin, amphetamine, benzodiazepines, cannabis, etc.), used in lateral flow immunoassays as revelation or detection molecules, with a focus on the components, the principles, the formats, and the mechanisms of reaction of these assays. Further, we report the monoclonal antibody advantages over the polyclonal ones used against drugs of abuse. The perspective on aptamer use for lateral flow assay development was also discussed as a possible alternative to antibodies in view of improving the limit of detection, sensitivity, and specificity of lateral flow assays.     

噻虫嗪单克隆抗体制备及酶联免疫吸附分析方法的建立

通过化学修饰合成了噻虫嗪人工半抗原,采用碳二亚胺法将该半抗原与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,成功制备了分子结合比合理的免疫原和包被原。经过免疫原免疫6周龄Balb/c小鼠、PEG介导免疫鼠脾细胞和骨髓瘤细胞的融合和阳性杂交瘤细胞的筛选和克隆化,获得了效价高达1∶6.4×105的抗噻虫嗪单克隆抗体,抗体亚类为IgG1型。优化了ELISA实验条件,建立了基于单克隆抗体技术的噻虫嗪残留间接竞争ELISA方法。本方法的抑制中浓度(IC50)为0.0255mg/L,检测灵敏度(IC20)为0.0022mg/L,检出限(IC10)为0.001mg/L。除噻虫胺外,该抗体与其它噻虫嗪结构类似物无交叉反应。以自来水为基质的噻虫嗪添加回收实验显示,0.01,0.5和10.0mg/L添加水平的回收率均大于75%,且各添加水平重复测定8次的相对标准偏差均小于8%,说明所建立的间接竞争ELISA准确度高,重复性好,适合水中噻虫嗪残留的检测。     

LC-MS/MS法定量测定人血浆中的氯氮平

建立了人血浆中氯氮平的固相萃取及LC-MS/MS定量检测方法.血浆中的氯氮平用固相萃取柱(Bond Elut Certify)萃取,采用Waters AtlantisTMd C18色谱柱,电喷雾离子源,正离子检测,多反应监测方式进行定量分析,SKF525a作为内标.对氯氮平的定量下限为100 ng/mL,氯氮平质量浓度...     

Epitope Mapping of Monoclonal Antibodies using Synthetic Oligosaccharides Uncovers Novel Aspects of Immune Recognition of the Psl Exopolysaccharide of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic Gram‐negative bacterium that can cause life‐threatening infections in critically ill and cystic fibrosis patients. The Psl exopolysaccharide of P. aeruginosa offers an attractive serotype‐independent antigen for the development of immunotherapies. Here, the first chemical synthesis of a panel of oligosaccharides derived from the exopolysaccharide of P. aeruginosa by a synthetic strategy that efficiently deals with the stereoselective installation of several β‐mannosides and the formation of a mannoside that is extended by saccharide moieties at C‐1, C‐2, and C‐3 in a crowded 1,2,3‐cis configuration is described. The approach was employed to prepare tetra‐, penta‐, and hexa‐ and decasaccharide part structures. The compounds were employed to define the epitope requirements of several functionally active monoclonal antibodies (mAbs) that can bind three distinct epitopes of Psl (class I, II, and III). The class II mAb reacted potently with each oligosaccharide indicating its epitope resides within the tetrasaccharide and does not require the branched mannoside of Psl. The class III antibody did not bind the tetra‐ or pentasaccharide; however, it did react potently with the hexasaccharide and weakly with the decasaccharide, suggesting a terminal glucoside is required for optimal binding. Unexpectedly, the class I mAb did not bind any of the oligosaccharides indicating that Psl contains a yet to be elucidated sub‐stoichiometric isoform. This study demonstrates that functional activity of a mAb does not only depend on the avidity of binding but also on the location of an epitope within a bacterial polysaccharide. The results also provide a strong impetus to analyze further the structure of Psl to identify the class I epitope, that is expected to provide an attractive target for the development of a synthetic vaccine for P. aeruginosa.     

固相萃取/液相色谱-串联质谱法测定鱼塘水中双甲脒及其代谢产物

建立了鱼塘水中双甲脒及其代谢产物的液相色谱-串联质谱检测方法。采用固相萃取前处理,考察了不同的固相萃取柱和洗脱溶剂对检测结果的影响,优化了固相萃取条件。结果表明,在优化条件下,4种目标物在20~2 000 ng/L范围内线性关系良好(r为0.997 5~0.999 1),该方法对双甲脒及其代谢产物的检出限为0.4~1.0 ng/L。鱼塘水中双甲脒及其代谢产物的回收率为69.7%~91.3%,方法具有良好的灵敏度、重现性、稳定性和专属性,可满足日常的分析要求。     

伏马菌素B1单克隆抗体制备及化学发光免疫检测方法的建立

制备了高亲和力的伏马菌素B₁（FB₁）单克隆抗体,进而建立了谷物中FB₁的直接竞争化学发光酶联免疫检测方法（dc-CLEIA）。采用碳化二亚胺法成功合成了FB₁人工抗原,经杂交瘤技术获得分泌FB₁特异性抗体的细胞株,高碘酸钠法酶标单克隆抗体后,建立了FB₁的dc-CLEIA,本方法的IC₅₀值为1.43μg/L,线性范围为0.32～8.40μg/L,检出限为0.13μg/L,竞争反应时间仅需20 min。玉米样品加标回收率为100.2%～115.4%,相对标准偏差小于8.7%,谷物样品的检测结果与高效液相色谱法及商业化ELISA试剂盒检测结果相符。     

Correlating Glycoforms of DC-SIGN with Stability Using a Combination of Enzymatic Digestion and Ion Mobility Mass Spectrometry

The immune scavenger protein DC-SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC-SIGN have not been reported. Herein, we report the development and application of a mass-spectrometry-based approach to both uncover and characterise the effects of O-glycans on the stability of DC-SIGN. We first quantify the Core 1 and 2 O-glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O-glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas-phase stability of the DC-SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC-SIGN is better correlated with the number of glycosylation sites.     

LC-MS/MS法定量测定人血浆中阿司匹林及其代谢产物水杨酸

阿司匹林是水杨酸类非甾体抗炎药,它能够快速而广泛地分布到人体多数组织和体液中,在体内水解产生水杨酸.     

Bead-Based Fluid Array Detection of Pentaerythritol Tetranitrate: Comparison of Monoclonal vs. Llama Polyclonal Antibodies

In today's security conscious environment rapid detection of explosives, such as pentaerythritol tetranitrate (PETN), the high energy material common in plastic explosives, is of critical importance. We evaluated two monoclonal antibodies and a rabbit polyclonal antibody, developed for the detection of PETN. Although these antibodies showed binding to bioconjugates of PETN as well as the hapten, PETN-succinate, we did not observe binding by free PETN. For demonstration of a competitive bead based fluid array immunoassay for PETN detection, we developed llama polyclonal antibody for the detection of free PETN. Our ultimate goal is developing anti-PETN single domain antibodies.     

酶切质谱法测定蛋白质的二硫键

二硫键是维持蛋白质活性构象的最重要因素之一,不正确的二硫键结构常导致蛋白质活性明显降低,甚至完全失活.早期测定二硫键主要采用"酶切-分离纯化-测序"法,过程繁琐,样品需求大.生物质谱为二硫键的测定提供了全新的策略.本文以重组人肝细胞生成素(rhHPO)--这一由我国独立发现和表达成功的、治疗严重肝病的潜在新药--为研究对象,探索了生物质谱进行二硫键定位的方法.     

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS

Intermediates of the purine biosynthesis pathway play key roles in cellular metabolism including nucleic acid synthesis and signal mediation. In addition, they are also of major interest to the biotechnological industry as several intermediates either possess flavor-enhancing characteristics or are applied in medical therapy. In this study, we have developed an analytical method for quantitation of 12 intermediates from the purine biosynthesis pathway including important nucleotides and their corresponding nucleosides and nucleobases. The approach comprised a single-step acidic extraction/quenching procedure, followed by quantitative electrospray LC-MS/MS analysis. The assay was validated in terms of accuracy, precision, reproducibility, and applicability for complex biological matrices. The method was subsequently applied for determination of free intracellular pool sizes of purine biosynthetic pathway intermediates in the two Gram-positive bacteria Corynebacterium glutamicum and Corynebacterium ammoniagenes. Importantly, no ion pair reagents were applied in this approach as usually required for liquid chromatography analysis of large classes of diverse metabolites.     

放射性核素标记单克隆抗体的化学研究进展

研究用放射性核素标记单克隆抗体作为肿瘤的导向药物,是核药物化学领域的研究热点.本文从化学角度介绍了单克隆抗体标记技术发展的概况,包括核素的选择,标记的方法,抗体的偶联修饰以及提高肿瘤/正常组织比的方法。