MALDI Q-TOF CID MS for Diagnostic Ion Screening of Human Milk Oligosaccharide Samples

Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.     

猪的遗传缺陷与异常

研究了耐盐与不耐盐番茄自交系在盐胁迫与正常栽培条件下根中蛋白质表达上的差异。耐盐番茄根系中蛋白质含量高于不耐盐番茄，正常栽培条件下番茄根系蛋白质含量高于盐处理番茄。蛋白质提取液不但在总蛋白质数量上有所不同，而且在蛋白质种类上亦有所差异。经过不同的双向凝胶电泳分离后，经过PD Quest软件和Q—TOF质谱分析结果为，在盐胁迫条件下共有8种蛋白质在表达上差异显著。磷酸葡萄糖脱氢酶、乙醇脱氢酶催化和3-异戊基苹果酸脱氢酶在盐处理的耐盐番茄根系中显著增加，异戊烯二磷酸(IDP)异构酶显著减少。     

奋乃静在人胆汁中的代谢物分析

建立了超高效液相色谱 四极杆串联飞行时间质谱（UPLC/Q-TOF MS）检测并表征奋乃静在人胆汁中代谢物的方法。T管收集一名精神病患者服用奋乃静后的胆汁样品,经乙腈沉淀蛋白预处理后,采用UPLC/Q-TOF MS进行分析。根据高分辨质谱给出的准确分子质量信息推测可能的分子式,结合MS E功能采集的前体离子和产物离子信息,利用质量缺损过滤（MDF）和generic dealkylation等代谢物鉴定软件筛选代谢物。通过对比原形药物和代谢物的质谱裂解途径,推测可能的代谢物结构。在服药（4 mg,b.i.d）后的人胆汁中,共检测到29种奋乃静代谢物,包括I相代谢物16种,II相代谢物13种,其中16种为首次报道的新颖代谢物。奋乃静在人体内的主要代谢途径包括羟基化、脱氢、N-去烷基化、甲基化、硫酸及葡萄糖醛酸结合等,该结果进一步完善了奋乃静在人体内的代谢途径。     

UPLC/Q-TOF MS/MS负离子模式下环氧烷型环烯醚萜苷的结构表征

利用四极杆飞行时间串联质谱(Q-TOF MS/MS)研究4个环氧烷型环烯醚萜苷同系组分(胡黄连苷Ⅰ、胡黄连苷Ⅱ、胡麻属苷、梓醇)的质谱裂解行为。在大气压化学电离源负离子(APCI~-)模式下,该类同系组分主要的裂解途径除了常见的母环上取代基的断裂,如中性丢失H_2O、CO_2和葡萄糖基等,生成~(1,6)F~-、~(1,4)F~-等特征碎片离子;另外还存在葡萄糖基环的断裂,生成~(0,4)A_1~-、~(1,4)A_1~-及~(2,4)A_1~-等碎片离子。通过对上述环氧烷型同系组分的质谱裂解行为进行归纳总结,依据其裂解规律,利用UPLC/Q-TOF MS/MS对胡黄连提取物中的环烯醚萜苷类化合物进行表征,共初步鉴定出10个环烯醚萜苷,其中包括8个环氧烷型环烯醚萜苷(胡黄连苷Ⅰ、胡黄连苷Ⅱ、胡黄连苷Ⅲ、胡黄连苷Ⅳ、黄金树苷、梓苷、婆婆纳苷和Piscroside B)和2个环戊烷型环烯醚萜(Boschnaloside和Mussaenosidic acid)。该方法有助于推动胡黄连在物质基础表征、质量控制和临床应用上的开发和利用。     

Isolation and structural characterisation of a propoxyphenyl-linked thiohomosildenafil analogue found in a herbal product

A propoxyphenyl-linked thiohomosildenafil analogue, one of the sildenafil analogues, was found in an herbal product. It was isolated by semi-preparative high-performance liquid chromatography (HPLC). The structure was established based on a comparison of chromatographic and spectroscopic behaviour with other sildenafil analogues using HPLC with diode array detection, quadrupole time-of-flight mass spectrometry (Q-TOF/MS), and nuclear magnetic resonance (NMR) spectroscopy. The HPLC analysis showed separation from known sildenafil analogues with a similar chromatographic retention time. An [M + H]⁺ ion at m/z 519.22 was detected by mass spectrometry corresponding to an empirical formula of C₂₄H₃₄N₆O₃S₂. The structure was similar to that of thiohomosildenafil, except that the ethoxy group attached to the phenyl ring was substituted for a propoxy group. It was assigned as 5-[2-propoxy-5-(4-ethylpiperazin-4-ylsulfonyl)phenyl]-3-methyl-1-n-propyl-4,5,dihydro-1H-pyrazole[7,1,d]pyrimidin-4-thione and named as propoxyphenyl-thiohomosildenafil because the structure was considerably similar to thiohomosildenafil.     

Surveillance of nitrite level in cubilose: Evaluation of removal method and proposed origin of contamination

The discovery of nitrite contamination aroused public concern on the safety of cubilose consumption. Here, forty-eight batches of cubilose were randomly purchased from Hong Kong market. The median nitrite content of different types of cubilose was as follows: Red cubilose (600 ppm) > Yellow cubilose (510 ppm) ;> White cubilose (100 ppm). Under a developed standardized processing method, up to 98% of nitrite was removed from cubilose; nitrite was not detected in the stewed cubilose. To search for the source of nitrite, droppings from swiftlets and water samples were collected from production sites of cubilose in Malaysia and Indonesia, and which contained a high amount of nitrate. On the other hand, the protein extract of cubilose was subjected to proteomics analysis. A microbial nitrate reductase was identified by mass spectroscopy, which converted nitrate to nitrite in cubilose. A specific inhibitor of nitrate reductase successfully abolished the nitrate reduction activity found in cubilose, which subsequently reduced the nitrite in cubilose. This phenomenon was successfully proven by the field study. Thus, the nitrite on cubilose could be a result of the contaminating nitrate reductase.     

应用超高效液相色谱-四级杆-飞行时间质谱 鉴定克伦特罗在猪尿中的主要代谢产物

目的 应用超高效液相色谱/四级杆-飞行时间质谱技术分析鉴定了克伦特罗在猪尿中的代谢产物,并推测克伦特罗在猪体内的主要代谢途径。方法 按10 mg/kg bw的剂量,给猪口服灌食克伦特罗,分别采集给药前及给药后的尿液样品。应用超高效液相色谱/四级杆-飞行时间质谱技术对样品进行分析,采用MetaboLynx XS软件进行数据处理,共检测到6 种克伦特罗的代谢产物,并根据碎片离子信息进行了结构鉴定。结果 猪尿中克伦特罗的代谢产物包括4-N-羟基克伦特罗（4-N-OH-CLE）、4-硝基克伦特罗（4-NO2-CLE）、克伦特罗及4-N-OH-CLE的葡萄糖醛酸结合物（GLU-CLE和GLU-OH-CLE）等。结论 根据所检测到的代谢产物,克伦特罗在猪体内的代谢途径包括4-N-氧化和葡萄糖醛酸结合等。     

Saturated aqueous ozone degradation of deoxynivalenol and its application in contaminated grains

Deoxynivalenol (DON) is a secondary metabolite produced by Fusarium graminearum in grains, food and contaminated feed, which can lead to many adverse health effects to humans and livestock. The degradation of DON in different contaminated grains oxidated by saturated aqueous ozone (80 mg L⁻¹) was monitored by ultra-high-performance liquid chromatography, tandem-quadrupole-detection mass spectrometry (UPLC–TQD-MS). Results suggest that ozone has a significant effect on DON reduction in solution. When 80 mg L⁻¹ gaseous ozone was used to treat 10 mg L⁻¹ of DON solution, the degradation rate of DON reached 83% within 7 min, while the respective detoxification rates of contaminated wheat, corn and bran by saturated aqueous ozone (80 mg L⁻¹) were 74.86%, 70.65% and 76.21 in 10 min. Ozone at 80 mg L⁻¹ was applied on various DON solution concentrations at 1 mg L⁻¹, 10 mg L⁻¹ and 20 mg L⁻¹ in ultra-pure water. In this paper, the degradation procedure for DON is calculated and described by a first-order kinetic equation. At lower levels (20 mg L⁻¹) of aqueous ozone, intermediate degradation products were observed by ultra-ne quickly and effectively degrades DON and toxicity in various contaminated grains in a matter of minutes. Therefore, ozonation is projected to be an effective, fast, and safe method for DON degradation.     

Alcalase-generated proteolysates of stone fish (Actinopyga lecanora) flesh as a new source of antioxidant peptides

In the present study, the alcalase-generated proteolysates obtained after 8 h of proteolysis of stone fish flesh showed the most potent antioxidant activity in terms of DPPH^? radical scavenging activity (77.43%, IC₅₀ of 0.5 mg/mL), ABTS^? radical scavenging activity (92.73%, IC₅₀ of 0.33 mg/mL) and FRAP value (39.2 mmol/100 mL FeSO₄). These proteolysates profiled and characterized as antioxidative peptides. The proteolysates were initially subjected to ultrafiltration using MWCO Spin-X UF. Potent fractions were further characterized based on hydrophobicity using reversed-phase high-performance liquid chromatography (RP-HPLC) and isoelectric point using an OFFGEL isoelectric focusing fractionator. Results indicated that most of the antioxidative peptides found in fractions with a molecular weight (MW) of less than 2 kDa, hydrophilic (hydrophilicity >80%) and basic (pI = 9.7). The final purified fraction with the highest antioxidant activity was selected for peptide identification and sequencing using Q-TOF mass spectrometry. A total of four peptides were identified, from which Peptide 1 (GVSGLHID) showed the highest antioxidant activity and this has potential as a novel bioingredient of nutraceuticals and functional foods to promote human health.