An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins

A sensitive, rapid, quantitative strategy has been developed for O-glycan analysis. A structural database has been constructed that currently contains analytical parameters for more than 50 glycans, enabling identification of O-glycans at the subpicomole level. The database contains the structure, molecular weight, and both normal and reversed-phase HPLC elution positions for each glycan. These observed parameters reflect the mass, three-dimensional shape, and hydrophobicity of the glycans and, therefore, provide information relating to linkage and arm specificity as well as monosaccharide composition. Initially the database was constructed by analyzing glycans released by mild hydrazinolysis from bovine serum fetuin, synthetic glycopeptides, human glycophorin A, and serum IgA1. The structures of the fluorescently labeled sugars were determined from a combination of HPLC data, mass spectrometric composition and mass fragmentation data, and exoglycosidase digestions. This approach was then applied to human neutrophil gelatinase B and secretory IgA, where 18 and 25 O-glycans were identified, respectively, and the parameters of these glycans were added to the database. This approach provides a basis for the analysis of subpicomole quantities of O-glycans from normal levels of natural glycoproteins.     

七种植物籽油中脂肪酸成分的比较分析(英文)

为寻求新的食用油资源,发展了一种快速可靠的气相色谱-质谱联用方法,用于植物籽油中脂肪酸成分的定性鉴定和含量测定。所建立的方法成功用于葡萄籽、南瓜籽和猕猴桃籽等七种植物籽油中的棕榈酸、十八烷酸、油酸、亚油酸和α-亚麻酸的定性定量分析。结果表明,刺葡萄籽油、普通葡萄籽油、国外葡萄籽油、南瓜籽油、枸杞籽油和西番莲籽油均具有相似的脂肪酸谱,尽管其中它们所含上述五种脂肪酸含量不同,由于均存在人体所必需的饱和与不饱和脂肪酸,故可以用作替代食用油。猕猴桃籽油因为其存在高含量的α-亚麻酸成分,可能是更好的食用油和营养油资源。本文首次对枸杞籽油、西番莲籽油和猕猴桃籽油脂肪酸成分进行绝对含量分析,为新的食用油资源的开发提供了重要的依据。     

基于GC-MS和UPLC-QTOF/MS技术的灵芝孢子粉化学成分分析

采用极性不同的6种溶剂（石油醚、乙酸乙酯、丙酮、乙醇、甲醇和水）、按索氏提取法逐级萃取破壁灵芝孢子粉,并同时运用气相色谱-质谱联用（GC/MS）和超高效液相串联四极杆飞行时间质谱（UPLC-QTOF/MS）技术对各萃取物进行化学成分分析与鉴定。结果表明：GC/MS共鉴定出101种化合物,其中酸类10种、酯类40种、醇类7种、酮类6种、酚类2种、烃类18种、甾类9种和杂原子化合物9种;UPLC-Q-TOF/MS共推断出40种化合物,其中倍半萜类1种、二萜类1种、三萜类9种、生物碱类4种、酰胺类7种、有机酸类9种以及其他化合物9种。两种测定方法间共有化合物仅1种,仅存在于5种有机溶剂（石油醚、乙酸乙酯、丙酮、乙醇和甲醇）萃取物之一的化合物共105种,2种或2种以上萃取物共有的化合物共31种,实验方法较好地实现了样品中化合物组分的充分分离,扩大了可检测化合物的范围。研究结果为灵芝孢子粉中化学成分的系统分析与鉴定、及灵芝孢子粉的化合物谱图库的完善提供了基础资料,为相关药理、药效分析及灵芝的药用模式真菌研究提供参考。     

Effects of feeding bile acids and a bile acid sequestrant on hepatic bile acid composition in mice

An improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was established for the simultaneous analysis of various bile acids (BA) and applied to investigate liver BA content in C57BL/6 mice fed 1% cholic acid (CA), 0.3% deoxycholic acid (DCA), 0.3% chenodeoxycholic acid (CDCA), 0.3% lithocholic acid (LCA), 3% ursodeoxycholic acid (UDCA), or 2% cholestyramine (resin). Results indicate that mice have a remarkable ability to maintain liver BA concentrations. The BA profiles in mouse livers were similar between CA and DCA feedings, as well as between CDCA and LCA feedings. The mRNA expression of Cytochrome P450 7a1 (Cyp7a1) was suppressed by all BA feedings, whereas Cyp7b1 was suppressed only by CA and UDCA feedings. Gender differences in liver BA composition were observed after feeding CA, DCA, CDCA, and LCA, but they were not prominent after feeding UDCA. Sulfation of CA and CDCA was found at the 7-OH position, and it was increased by feeding CA or CDCA more in male than female mice. In contrast, sulfation of LCA and taurolithocholic acid (TLCA) was female-predominant, and it was increased by feeding UDCA and LCA. In summary, the present systematic study on BA metabolism in mice will aid in interpreting BA-mediated gene regulation and hepatotoxicity.     

Rapid analysis of naphthalene and its metabolites in biological systems: Determination by high-performance liquid chromatography/fluorescence detection and by plasma desorption/chemical ionizations mass spectometry

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.     

Comparison of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods to quantify alpha-tocopherol and alpha-tocopherolquinone levels in human plasma.

Two mass spectrometric methods were established for the quantitative analyses of alpha-tocopherol (TH) and its oxidation product alpha-tocopherolquinone (TQ) in human plasma. Both methods make use of isotopically labeled internal standards of different levels of deuteration (d3-TH and d6-TQ). Plasma (100 microl) was saponified in the presence of a mixture of antioxidants, and then TH and TQ were extracted with hexane. With the GC-MS method, the analytes were first converted into O-trimethylsilyl derivatives before analysis in the selective ion monitoring mode. The derivatization procedure led to the quantitative conversion of TQ into the O-trimethylsilyl derivative of tocopherolhydroquinone, giving rise to a more stable molecule with less fragmentation than for TQ. The increased stability of the molecule resulted in an enhanced contribution of the base peak to the total observed ions and therefore an increased sensitivity of the base peak for quantification. With the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, TH and TQ were detected by multiple reaction monitoring after positive electrospray ionization. The GC-MS and LC-MS/MS methods showed nearly the same accuracy (>95%) and the same within-day precisions, with less than 5 and 10% for TH and TQ, respectively. The between-day precision and the limit of quantification for TQ in plasma were better by LC-MS/MS (4%; 3 nM) than by GC-MS (21%; 10 nM). Analysis and method validation were carried out with plasma samples obtained from a male volunteer pre- and postexercise. Both techniques showed that the ratio of TQ/TH was elevated by 35% immediately after exercise and had returned to basal levels when measured 24 h later.     

One-pot microwave derivatization of target compounds relevant to metabolomics with comprehensive two-dimensional gas chromatography

Metabolomics has been defined as the quantitative measurement of all low molecular weight metabolites (sugars, amino acids, organic acids, fatty acids and others) in an organism's cells at a specified time under specific environmental/biological conditions. Currently, there is considerable interest in developing a single method of derivatization and separation that satisfies the needs for metabolite analysis while recognizing the many chemical classes that constitute the metabolome. Chemical derivatization considerably increases the sensitivity and specificity of gas chromatography–mass spectrometry for compounds that are polar and have derivatizable groups. Microwave-assisted derivatization (MAD) of a set of standards spanning a wide range of metabolites of interest demonstrates the potential of MAD for metabolic profiling. A final protocol of 150 W power for 90 s was selected as the derivatization condition, based upon the study of each chemical class. A study of the generation of partially derivatized components established the conditions where this could potentially be a problem; the use of greater volumes of reagent ensured this would not arise. All compounds analyzed by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry in a standard mixture showed good area ratio reproducibility against a naphthalene internal standard (RSD < 10% in all but one case). Concentrations tested ranged from 1 μg/mL to 1000 μg/mL, and the calibration curves for the standard mixtures were satisfactory with regression coefficients generally better than 0.998. The application to gas chromatography–quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry for a typical reference standard of relevance to metabolomics is demonstrated.     

Protein-free models for the proton-coupled reduction of haemoproteins

Reduction of the bis-pilocarpate-haemin complex at pH greater than or equal to 10 involves the simultaneous uptake of an electron by the Fe(III) ion and a proton by the pendant alkoxide group of an axial ligand. This provides a protein-free model for reactions such as the proton-coupled reduction of cytochromes which involve cooperative Coulombic interaction between two non-bonded sites.     

Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples

High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-peak with reliable retention time (Rt.). However, in the current study, we have found that under the same LC-MS conditions, the Rt. and shape of LC-peaks of bile acids in urine samples from animals fed dissimilar diets differed significantly among each other. To verify this matrix effect, 17 authentic bile acid standards were dissolved in pure methanol or in methanol containing extracts of urine from pigs consuming either breast milk or infant formula and analyzed by LC-MS/MS. The matrix components in urine from piglets fed formula significantly reduced the LC-peak Rt. and areas of bile acids. This is the first characterization of this matrix effect on Rt. in the literature. Moreover, the matrix effect resulted in an unexpected LC behavior: one single compound yielded two LC-peaks, which broke the rule of one LC-peak for one compound. The three bile acid standards which exhibited this unconventional LC behavior were chenodeoxycholic acid, deoxycholic acid, and glycocholic acid. One possible explanation for this effect is that some matrix components may have loosely bonded to analytes, which changed the time analytes were retained on a chromatography column and interfered with the ionization of analytes in the MS ion source to alter the peak area. This study indicates that a comprehensive understanding of matrix effects is needed towards improving the use of HPLC and LC-MS/MS techniques for qualitative and quantitative analyses of analytes in pharmacokinetics, proteomics/metabolomics, drug development, and sports drug testing, especially when LC-MS/MS data are analyzed by automation software where identification of an analyte is based on its exact molecular weight and Rt.     

Liquid chromatography “on-flow” 1H nuclear magnetic resonance on native glycosphingolipid mixtures together with gas chromatography/mass spectrometry on the released oligosaccharides for screening and characterisation of carbohydrate-based antigens from pig lungs

Glycosphingolipids were prepared from pig lung and pooled into two fractions with (i) 3 sugar residues, and (ii) 3 sugar residues. Oligosaccharides were prepared and used for gas chromatography, gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The glycolipid fractions i and ii were further characterised and purified using a novel method based on high performance liquid chromatography on-flow proton nuclear magnetic resonance. The LC on-flow NMR technique showed good chromatographic separation and gave NMR spectral information which could be used as guidance for pooling of the separated mixture glycolipids. Conventional ¹H NMR, thin layer immunostaining, gas chromatography, gas chromatography/mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry were used to characterise the glycolipids and to validate LC-NMR spectral data.