基于SWATH定量质谱技术的肥胖小鼠脂肪线粒体蛋白质组分析

目的揭示肥胖诱发脂肪组织线粒体蛋白质组的变化,并探索变化的可能机制,实现针对小鼠脂肪组织线粒体蛋白质组的深度测序和准确定量。方法利用数据依赖型(DDA)质谱方法鉴定小鼠白色脂肪组织和棕色脂肪组织中的线粒体蛋白;利用新型SWATH(sequential windowed acquisition of all theoretical mass spectra)质谱技术定量肥胖小鼠和野生型小鼠不同脂肪组织中线粒体蛋白,同时对表达差异的线粒体蛋白进行功能分析。实验步骤:1用组织线粒体提取试剂盒特异性提取不同类型脂肪组织线粒体蛋白;2用10%聚丙烯酰胺凝胶电泳预分离线粒体蛋白;3利用DDA方法建立线粒体蛋白质谱文库;4利用SWATH质谱方法定量线粒体蛋白;5利用Peak View2.0软件提取碎片离子色谱峰并进行积分计算其峰面积;6根据KEGG(kyoto encyclopedia of genes and genomes)通路和GO(Gene Ontology)功能分类分析肥胖小鼠和正常小鼠脂肪组织表达差异蛋白的生物功能。结果在白色脂肪组织和棕色脂肪组织分别准确鉴定并定量线粒体定位蛋白1000和1039种,3次生物学重复实验中共鉴定包括线粒体氨肟还原成分1在内的25种线粒体蛋白在白色脂肪组织特异性表达,包括非偶联蛋白3在内的21种线粒体蛋白在棕色脂肪组织特异性表达。肥胖小鼠与正常小鼠相比,白色脂肪组织中共有25种线粒体蛋白表达显著上调(上调倍数≥2,P<0.01),有47种线粒体蛋白表达显著下调(下调倍数≥2,P<0.01);棕色脂肪组织中有26种线粒体蛋白表达显著上调(上调倍数≥2,P<0.01),有21种线粒体蛋白表达显著下调(下调倍数≥2,P<0.01)。结论脂肪组织线粒体蛋白质组的定量及生物信息学的分析表明,肥胖导致线粒体内三羧酸循环、氧化磷酸化、脂肪酸的合成和降解以及对外源性物质的代谢等重要的信号通路发生紊乱,并准确鉴定、定量其相关表达差异蛋白,为肥胖导致代谢紊乱的机制研究提供重要的数据支持。     

阿尔茨海默病模型小鼠脑组织生物标记物代谢组学分析

目的研究阿尔茨海默病(alzheimer's disease,AD)小鼠脑组织UPLC-MS/MS代谢物谱的变化,确定AD相关的生物标记物。方法小鼠单侧海马注射β淀粉样蛋白1-42(Aβ1-42)建立AD模型,采用Morris水迷宫试验和免疫组织化学法分别检测各组小鼠学习记忆能力和脑组织的病理学变化,采用超高效液相色谱-串联质谱(UPLC-MS/MS)技术分析测定正常组、AD模型组小鼠脑组织匀浆的代谢物谱图,数据导入masslynx软件进行主成分分析(principal component analysis,PCA)来检测脑组织中代谢产物的变化。结果经PCA分析,得分图显示正常对照组和AD模型组明显分离。确定十六碳神经鞘氨醇、二氢神经鞘氨醇、4-羟神经鞘氨醇、溶血磷脂酰胆碱C 20∶4、溶血磷脂酰胆碱C18∶3、溶血磷脂酰胆碱C 16∶0、溶血磷脂酰胆碱C 13∶0、次黄嘌呤、神经酰胺N 40∶1共9个潜在的生物标志物。与正常对照组相比,AD小鼠脑组织中鞘氨醇、溶血磷脂酰胆碱的含量明显降低(P<0.05);次黄嘌呤,神经酰胺N40∶1含量明显升高(P<0.05)。结论 AD模型小鼠脑组织的代谢物谱发生了明显改变,为AD的早期诊断和治疗提供科学依据。     

非标记定量蛋白质组方法分析牡蛎多糖抗D-半乳糖导致的小鼠衰老作用的研究

目的 研究牡蛎多糖抗衰老作用机制。方法 利用非标记定量蛋白质组方法分析牡蛎多糖抗衰老作用机制。将去除高丰度蛋白后的小鼠血清样本经酶解后进行LC-MS/MS分析,利用Maxquant软件(版本号1.3.0.5)查库,进行LFQ非标定量分析。结果 本研究共鉴定到4624个肽段,541个蛋白质。将模型组与牡蛎多糖治疗组进行t-test分析,得到20个差异表达蛋白质。对在小鼠数据库中查找到的19个差异蛋白质进行分析,发现这些差异蛋白质参与15个生物过程、具有8类分子功能、存在于5个细胞组分中(Level 2)。这些差异表达蛋白质中,peroxiredoxin-2(AC：Q61171)和malate dehydrogenase(AC：P14152)的上调以及xanthine dehydrogenase/oxidase(AC：Q00519)的下调可以提高机体清除自由基的能力。结论 牡蛎多糖是通过提高机体清除自由基的能力来发挥其抗衰老作用的。     

生物质谱技术的发展及在蛋白质结构研究中的应用

综述生物质谱技术的发展现状，包括电喷雾电离质谱、基质辅助激光解吸电离质谱、串联质谱、电喷雾解吸电离质谱、表面增强激光解吸电离-飞行时间质谱等，以及这些技术在蛋白质结构研究中的应用进展。生物质谱技术已成为蛋白质结构研究的重要工具。     

骨髓间充质干细胞来源外泌体对炎症微环境中巨噬细胞表型及软骨细胞的调控作用

目的 研究骨髓间充质干细胞来源外泌体（BMSCs-Exo）在炎症微环境中对巨噬细胞表型极化及软骨修复的调控作用。方法 差速离心法提取BMSCs-Exo,透射电镜观察外泌体形态,Western blot检测Alix、TSG101表达;使用不同浓度的BMSCs-Exo（10 μg/mL 和50 μg/mL）处理M1巨噬细胞及IL-1β刺激的软骨细胞,CCK-8测定BMSCs-Exo促巨噬细胞增殖能力,Western blot和qRT-PCR验证BMSCs-Exo对M1型巨噬细胞表型极化的影响,炎症相关基因IL-1β、IL-6的表达水平,以及成软骨相关因子COL II、MMP-13、TGF-β1的表达水平。结果 BMSCs-Exo为圆形双层囊泡,表达Alix、TSG101,可促进巨噬细胞增殖。与对照组比较,M1型巨噬细胞经BMSCs-Exo处理后,M1型极化标记物（iNOS）的表达水平降低,M2型极化标记物（Arg-1）的表达水平升高（P<0.05）;炎症相关基因IL-1β、IL-6的表达水平显著降低（P<0.05）,炎症软骨细胞中的COL II、TGF-β1表达升高,MMP-13表达水平降低（P<0.05）。结论 BMSCs-Exo可调控M1型巨噬细胞向M2型巨噬细胞极化,降低炎症因子表达水平,促进炎症微环境中软骨修复。     

氚标记的Bcl-2反义核酸(F951)在小鼠体内的药代动力学实验研究

目的　研究一种新的氚标记的Bcl 2反义寡核苷酸 3H F95 1单次静脉注射后在小鼠体内的药代动力学过程。方法　用氚标记F95 1,用液闪仪检测放射性浓度 ,用 3P87软件判断房室模型 ,计算各种参数。用液闪仪检测3H F95 1在小鼠体内各器官中的分布浓度 ,检测给药后 72h内药物从小鼠尿和粪中排泄的量。结果　3H F95 1单次静脉注射后在小鼠体内的动力学过程符合二室模型 ,3种剂量时主要的参数血浆分布半衰期 (T1/ 2α)在 10～ 15min之间 ,消除半衰期 (T1/ 2 β)在 2～ 4 5h之间。3H F95 1在肾、肝、脾、骨髓中分布浓度高于其它器官 ,在心、肺、脑、肠、皮肤、脂肪、生殖腺中也有低水平的分布。3H F95 1主要经肾从尿中排泄 ,给药后 2 4h内的累积排泄量占给药量的 5 0 47%。3H F95 1从粪中排泄的量甚微。结论　3H F95 1在小鼠体内药代动力学过程的研究为其进一步的开发具有指导价值     

小鼠血管内皮细胞的培养、鉴定及巨噬细胞移动抑制因子对其促增殖作用的研究

目的探讨小鼠血管内皮细胞的培养、鉴定方法及巨噬细胞移动抑制因子(MIF)对其增殖活性的调节作用。方法取小鼠主动脉,去除周围的脂肪和结缔组织,将动脉环剪成0.5 mm大小,放入基质胶处理的12孔培养板内,待细胞长满,用胰酶消化后,取单细胞悬液用抗CD146免疫磁珠纯化,再用CD31抗体鉴定细胞纯度,按104个细胞/孔加入96孔板培养,细胞70%～80%融合时用不同浓度MIF或MIF抑制剂处理细胞,48 h后用甲基噻唑基四唑(MTT)检测细胞增殖程度。结果经CD146磁珠纯化后细胞纯度为95%以上,MTT检测结果显示低剂量MIF能促进血管内皮细胞的增殖,并具有剂量效应关系,而高剂量MIF则与MIF抑制剂一样,抑制血管内皮细胞的增殖。结论成功建立了简单可行的小鼠血管内皮细胞的体外培养方法,并证实一定剂量范围内的MIF能促进血管内皮细胞的增殖,说明MIF在血管内皮细胞的多种病理生理反应中起重要作用。     

Slit分泌蛋白家族2/Slit分泌蛋白家族特异性受体1在骨髓增生异常综合征中的表达及意义

目的 探讨Slit分泌蛋白家族2/Slit分泌蛋白家族特异性受体1(Slit2/Robo1)在骨髓增生异常综合征(MDS)中的表达及临床意义.方法 以2017年8月至2019年6月就诊于徐州医科大学附属医院的51例初诊MDS病人为研究对象,采用Ficoll密度梯度离心法分离51例MDS病人治疗前后骨髓单个核细胞,并通过...     

基于液质联用技术的代谢组学生物样本——血液及尿液的收集和处理

代谢组学是定性及定量描述内源性代谢物变化的科学,正广泛应用于疾病诊断和预后过程中。血液和尿液是代谢组学研究中常用的生物样本,但因其代谢物繁多、成分复杂,亟须使用灵敏度高、特异性强的分离分析技术。液相色谱-质谱联用（liquid chromatography-mass spectrometry, LC-MS）技术分析速度快、分离效率高,非常适用于代谢组学等方面的研究。生物样本的采集、处理过程是实验中最关键的一步,直接决定着实验结果的准确性,因此生物样本预处理过程的标准化显得尤为重要。本文综述了常用的生物流体—血液和尿液的收集、处理及储存方法,以期为有关代谢组学研究提供参考。     

玉米须提取物的小鼠急性毒性实验及降血糖作用观察

目的:研究玉米须乙醇及水提取物灌胃给药对小鼠的急性毒性作用,并观察其降血糖作用.方法:改良寇氏法计算玉米须提取物半数致死量(LD50)及其95％可信区间(CI).观察玉米须提取物对正常小鼠、葡萄糖致高血糖模型小鼠、肾上腺素致血糖升高小鼠的降血糖作用.结果:玉米须乙醇提取物LD50=203.106 g/kg(95％ CI 188.665～218.652 g/kg);水提取物LD50=105.203 g/kg(95％ CI 95.249～116.197 g/kg).玉米须水提取物和乙醇提取物对正常小鼠血糖无影响;乙醇提取物30 g/kg可明显降低葡萄糖导致的小鼠血糖升高(P＜0.01);水提取物与乙醇提取物30 g/kg可明显降低肾上腺素致小鼠升高的血糖值(P＜0.05或P＜0.01),且乙醇提取物可明显升高高血糖小鼠的肝糖原含量(P＜0.01).结论:玉米须水和乙醇提取物为无毒级中药提取物.两种提取物对正常小鼠无降低血糖作用,对高糖和肾上腺素所致的小鼠高血糖有降低作用,同时具有促进糖异生,调节糖代谢的作用.     

植物雌激素金雀异黄酮通过p38MAPK通路促进骨髓间充质干细胞向成骨细胞分化

目的研究丝裂原激活的蛋白激酶(m itogen-activatedprote in k inases,MAPKs)的亚型p38 MAPK在植物雌激素金雀异黄酮(Gen iste in)促进小鼠骨髓间充质干细胞(bone m ar-row-derived m esenchym al stem cells,BMSCs)向成骨细胞分化过程中的作用。方法BMSCs用无酚红-αMEM(含经活性碳吸附的10%FBS、β-磷酸甘油、维生素C)培养,先用Gen iste in处理细胞,观察BMSCs向成骨细胞分化情况,然后用SB203580(p38 MAPK通路阻断剂)以及PD98059(p44/42MAPK通路阻断剂)阻断相应的通路后,再用Gen iste in处理细胞,观察BMSCs向成骨细胞分化情况,并同时观察上述处理后MAPK通路的变化。测定碱性磷酸酶(ALP)活性和钙(Ca)沉积量反映BMSCs向成骨细胞分化状况,用W esternb lot来检测MAPK通路是否激活。结果Gen iste in(0.01,0.1,1μmol.L-1)剂量依赖性增加小鼠BMSCs细胞内ALP活性和细胞外Ca沉积量,并同时引起p38MAPK通路的激活和p44/42MAPK通路的抑制。SB203580预处理能减弱Gen iste in刺激引起的p38MAPK通路的激活并同时阻止Gen iste in诱导的BMSCs向成骨细胞分化。结论Gen iste in在0.01~1μmol.L-1剂量范围内可通过p38MAPK通路促进小鼠BMSCs向成骨细胞分化。     

The characterization of isobaric product ions of fentanyl using multi‐stage mass spectrometry,high‐resolution mass spectrometry and isotopic labeling

This study uses a combination of multi‐stage mass spectrometry (MSⁿ), accurate mass measurements – with high‐resolution mass spectrometry (HRMS) – and isotopic labeling to characterize the fragmentation behavior of fentanyl and 4‐ANPP. By understanding the fragmentation behavior of fentanyl and its analogs in more detail, toxicologists and seized drug analysts will be better poised to identify new and emerging fentalogs, which are increasingly common and deadly adulterants in the growing opioid crisis. Throughout the literature the product ion at m/z 188 is often the most abundant fragment in the mass spectrometric analysis of fentanyl and fentanyl analogs, and this fragment is used for both qualitative and quantitative determinations. Our work shows there are at least three different structures for the isobaric fentanyl product ions at m/z 188, and they each form and fragment via different pathways. The development of fragmentation mechanisms to explain the observed fragmentation pathways of fentanyl and its main precursor 4‐ANPP helps contribute to the advancement of knowledge about fentanyl fragmentation and could provide important information for the identification of future fentanyl analogs.     

Identification of alcohol‐binding site(s) in proteins using diazirine‐based photoaffinity labeling and mass spectrometry

Defining molecular targets of alcohol and understanding the molecular mechanism of alcohol actions are necessary to develop effective therapeutics for alcohol use disorder (AUD). Here, we describe a detailed protocol for identifying alcohol‐binding site(s) in proteins using diazirine‐based azialcohol as photoaffinity labeling agents. Upon photoirradiation, azialcohol photoincorporates into alcohol‐binding proteins. The stoichiometry and site of azialcohol photoincorporation can be determined using high‐resolution mass spectrometry. Identification of the alcohol‐binding residues in protein followed by measuring the biological significance of these residues in regulating alcohol action are important steps in characterizing the molecular targets of alcohol.     

5-Azacytidine inhibits proliferation and induces apoptosis of mouse bone marrow-derived mesenchymal stem cells

Abstract

In this study, we investigated the effect of 5-azacytidine on proliferation and apoptosis of bmMSCs. After exposure to different concentrations of 5-azacytindine, bmMSC proliferation and apoptosis were measured by MTT assay, Caspase-3 staining and western blotting. Our results show that 1?μM 5-azacytidine has no significant effect on viability of bmMSCs; however, 5–20?μM 5-azacytindine significantly inhibits bmMSC proliferation and expression of phospho-Akt; 10 and 20?μM 5-azacytidine markedly increases bmMSC apoptosis and expression of Caspase-3, Annexin V and Bax, and decreases expression of Bcl2. Our findings indicate that 5-azacytidine at the commonly used doses has toxicity to bmMSCs.     

克罗米酚对骨髓间质干细胞增殖和向成骨细胞分化的影响

目的　在离体条件下研究克罗米酚(CLO)对小鼠骨髓间质干细胞(BMSCs)增殖和向成骨细胞分化的影响。方法　在含经活性炭吸附的 10%血清的无酚红α MEM中加入β 磷酸甘油和维生素C诱导小鼠BMSCs向成骨细胞分化,同时加入 0 1nmol·L-1至 10nmol·L-1CLO处理细胞。用细胞计数来反映细胞增殖情况;测定碱性磷酸酶 (ALP)活性与钙的沉积量反映细胞向成骨细胞分化状态;用试剂盒来检测一氧化氮 (NO)的产物。结果　CLO ( 0 1 ~10nmol·L-1 )呈剂量依赖性地增加小鼠BMSCs的数目,ALP活性和钙的沉积量,同时培养基中NO代谢产物明显增加。分别加入雌激素受体 (ER)拮抗剂ICI182, 780 (0 1μmol·L-1 )及一氧化氮合酶抑制剂L NAME( 6mmol·L-1 )均可阻止CLO促进BMSCs的增殖及向成骨细胞分化的作用,并取消NO代谢产物的增加。结论　CLO在 0 1 ~10nmol·L-1剂量范围内具有雌激素样受体激活效应,可通过ER/NO途径促进小鼠骨髓间质干细胞的增殖及向成骨细胞分化。     

The proteomic analysis of human neonatal umbilical cord serum by mass spectrometry

Aim:

To investigate the proteome composition and function of human neonatal arterial umbilical cord.

Methods:

Serum proteomic analyses were performed on samples from both males and females by using a combination of techniques: (1) removal of six high-abundance proteins, (2) tryptic digestion of low-abundance proteins, (3) separation of peptide mixtures by reverse-phase high-performance liquid chromatography (RP-HPLC), and (4) peptide identification using electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Results:

A total of 837 non-redundant proteins were identified, with 213 male-specific and 239 female-specific proteins. Among them, 319 proteins were identified by at least 2 distinct peptides. The subcellular localization, function, and pathway involvement for each of the identified proteins were analyzed. A comparison of this neonatal proteome to that of adult serum proteome revealed novel biomarkers, such as alpha-fetoprotein and periostin that were specific to newborn infants.

Conclusion:

These data will contribute to a better understanding of the composition of umbilical cord serum and aid the discovery of novel biomarkers for the prenatal diagnosis of fetal abnormalities.     

Behaviour and ultrastructure of human bone marrow-derived mesenchymal stem cells immobilised in alginate-poly-l-lysine-alginate microcapsules

Abstract

Context: Human bone marrow mesenchymal stem cells (hBM-MSCs) show a great promise for the treatment of a variety of diseases. Despite the previous trials to encapsulate hBM-MSCs in alginate-poly-l-lysine-alginate (APA) systems, the various changes that follow immobilisation have not been ascertained yet. Objective: Determine the various consequences derived from entrapment on cell behaviour, putting special emphasis on the ultrastructure. Methods: hBM-MSCs were immobilised in APA microcapsules to further characterise their viability, metabolic activity, proliferation, VEGF-secretability, and morphology. Results: The VEGF produced by monolayer hBM-MSCs increased significantly 1 d post-encapsulation, and was maintained for at least 4 weeks. TEM imaging of cells revealed well preserved ultrastructure indicating protein synthesis and high metabolic activity. Conclusion: Although APA microencapsulation did not support 100% of fully viable hBM-MSCs for long-term cultures, it was conceived to enhance both VEGF secretion and metabolic activity while not losing their stemness characteristics.     

Determination of chlorpromazine in porcine muscle using high performance liquid chromatography-tandem mass spectrometry

A rapid and accurate high performance liquid chromatography tandem mass spectrometry (HPLS-MS) method was established for quantification of chlorpromazine in pork. The porcine samples were pretreated with acetonitrile to precipitate proteins and followed by extraction with tert-butyl methyl ether (TBME). The separation was performed on a 5 µm Agilent XDB-C₁₈ column with gradient elution. The mobile phase A was 0.01 mol/Lammonium formate in water and mobile phase B was acetonitrile. The flow rate was at 0.8 mL/min. Quantification was performed on a triple-quadrupole tandem mass spectrometer using the multiple selected reaction monitoring (MRM) mode. Transition of m/z +319.1 to 58.1 was used for the quantification of chlorpromazine. The method was validated at the concentration range of 0.4040 µg/kgto 8.080 µg/kg for chlorpromazine with correlation coefficient of 0.9999. The spiked recoveries were more than 80.0% and the limit of detection (LOD) was 0.052 µg/kg.The developed method, which offers advantages of convenience, rapid, specificity and higher sensitivity, can be used for determination of chlorpromazine hydrochloride in porcine muscles.     

Fast atom bombardment mass spectrometry of ceruletide and [Tyr 4] ceruletide

Fast atom bombardment (FAB) mass spectrometry has revealed a very useful technique in obtaining the mass spectrum of ceruletide (formerly caerulein), a decapeptide with a tyrosyl-O-sulphate residue, and of its desulphated analog. Molecular weight value for ceruletide is obtained only in the negative ion mode, whereas for [Tyr⁴] ceruletide this information is obtained both in negative and in positive ion detection. Positive ion mass spectra show more extended fragmentation which allows the complete amino acids sequence to be checked. The lability of the sulphate group has been confirmed, examining other sulphated peptides by field desorption mass spectrometry.     

The influence of chemical modifications on the fragmentation behavior of fentanyl and fentanyl‐related compounds in electrospray ionization tandem mass spectrometry

Fentanyl is a synthetic opioid that has been approved by the FDA as a general anesthetic because of its rapid onset and high potency. However, since 2013 an opioid epidemic involving fentanyl or fentanyl‐related compounds (FRCs) has swept the United States and caused numerous deaths in every state. The identification of novel FRCs is complicated by the rapid turnover of modifications to the core fentanyl structure. In this study, a series of 16 FRCs were analyzed using electrospray ionization tandem mass spectrometry (ESI‐MS/MS) to gain a deeper understanding of the conserved and unique fragmentation behaviors associated with substitution to the core fentanyl structure. This work provides an approach, based on the product ions from ESI‐MS/MS, to identify the modification site(s) on the core fentanyl structure for FRCs. Five common locations of substitution to the core fentanyl structure were used to assess the effect of substitution on the fragmentation behavior of FRCs. The proposed fragmentation pathways are supported through the combination of isotopic labeling, multi‐stage mass spectrometry (MSⁿ), and accurate mass measurements with high‐resolution mass spectrometry (HRMS). The identification of primary product ions specific to regions of substitution provides an additional tool for the identification of the location of substitution to the core fentanyl structure, which ultimately will assist toxicologists and seized drug analysts in the identification of emerging FRCs.