基于空气动力辅助离子化-超高分辨质谱成像技术的大鼠肾脏组织中多种类代谢物的分布研究
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摘要
质谱成像是近年来发展迅速的新型分子成像技术,在生物医药领域受到越来越广泛的关注。本研究采用敞开式的空气动力辅助离子化-高分辨质谱技术,建立了大鼠肾脏组织中内源性代谢物的质谱成像分析方法(AFAI-MSI)。大鼠肾脏组织经冷冻处理后制备冷冻组织切片,以乙腈-异丙醇-水(4∶4∶2,V/V)为喷雾溶剂,流速为5μL/min,喷雾气(N_2)压力为0.6 MPa,空气辅助气流速为45 L/min,质谱扫描范围为70~1000 Da,质量分辨率为70000。采用正离子检测模式的AFAI-MSI方法对大鼠肾脏组织切片进行了成像分析,结果发现有机胺、糖、神经递质、维生素、多肽、有机酸、甘油磷脂、鞘脂、甘油脂、固醇酯等38种不同类型、其含量差异达4个数量级的内源性代谢物,并观察到这些代谢物在肾脏中呈组织特异性分布,直观地呈现了与肾脏渗透压梯度形成有关的胆碱、乙酰胆碱、甜菜碱、磷酸胆碱和甘油磷酸胆碱等多种小分子代谢物的皮质-髓质轴向分布特征。上述结果表明,基于超高分辨质谱的AFAI-MSI分析方法无需样品预处理,灵敏度高,代谢物覆盖范围宽,可同时获取多种小分子代谢物的结构、含量及其空间分布信息,有望为肾脏中内源性代谢物的原位表征和代谢调控机制研究提供一种新的分析方法。
As a promising new molecular imaging technique,mass spectrometry imaging( MSI) has attracted more and more attention in the field of biomedicine. A method of air flow assisted ionization-ultra high resolution mass spectrometry-based mass spectrometric imaging( AFAI-MSI) was developed to profile endogenous metabolites in rat kidney tissue in this study. Rat kidneys were collected and cut into frozen tissue sections,and then were analyzed on an AFAI-MSI system in positive ion mode using acetonitrile-isopmpyl alcohol-water( 4∶ 4∶ 2,V/V,5 μL/min) as spray solvent,N2 as spray gas( 0.6 MPa) and air as assisting gas( 45 L/min). The mass range and resolution were set to be 70-1000 Da and 70000,respectively. As a result,a total of 38 metabolites,including organic amines,sugars,vitamins,peptides,neurotransmitters,organic acids,phospholipids, sphingolipids, glyceride, and cholesterol esters, were identified and imaged to characterize their tissue-specific distribution in kidney tissues, and some metabolites, such as choline,acetylcoline,betaine,phoshocholine,and glycerophosphocholine were found to have distinct distribution along the cortex-medulla axis,which may be involved in the formation of osmotic pressure gradient in the kidney.The proposed ultra high resolution mass spectrometry based AFAI-MSI method could work without sample pretreatment,showed high sensitivity and wide metabolite coverage,and was expected to provide a new analytical approach for the research of in situ characterization and metabolic regulation mechanism of endogenous metabolites in kidney.
As a promising new molecular imaging technique,mass spectrometry imaging( MSI) has attracted more and more attention in the field of biomedicine. A method of air flow assisted ionization-ultra high resolution mass spectrometry-based mass spectrometric imaging( AFAI-MSI) was developed to profile endogenous metabolites in rat kidney tissue in this study. Rat kidneys were collected and cut into frozen tissue sections,and then were analyzed on an AFAI-MSI system in positive ion mode using acetonitrile-isopmpyl alcohol-water( 4∶ 4∶ 2,V/V,5 μL/min) as spray solvent,N2 as spray gas( 0.6 MPa) and air as assisting gas( 45 L/min). The mass range and resolution were set to be 70-1000 Da and 70000,respectively. As a result,a total of 38 metabolites,including organic amines,sugars,vitamins,peptides,neurotransmitters,organic acids,phospholipids, sphingolipids, glyceride, and cholesterol esters, were identified and imaged to characterize their tissue-specific distribution in kidney tissues, and some metabolites, such as choline,acetylcoline,betaine,phoshocholine,and glycerophosphocholine were found to have distinct distribution along the cortex-medulla axis,which may be involved in the formation of osmotic pressure gradient in the kidney.The proposed ultra high resolution mass spectrometry based AFAI-MSI method could work without sample pretreatment,showed high sensitivity and wide metabolite coverage,and was expected to provide a new analytical approach for the research of in situ characterization and metabolic regulation mechanism of endogenous metabolites in kidney.
引文
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2 LIU Hui,CHEN Guo-Qiang,WANG Yan-Ying,LI Zhi-Li.Chinese J.Anal.Chem.,2011,39(1):87-90刘辉,陈国强,王艳英,李智立.分析化学,2011,39(1):87-90
3 Weiss R H,Kim K.Nat.Rev.Nephrol.,2012,8(1):22-33
4 Zhao Y Y.Clin.Chim.Acta,2013,422:59-69
5 Lalowski M,Magni F,Mainini V,Gotsopoulos A,Soliymani R,Chinello C,Baumann M.Nephrol.Dial.Transplant.,2013,28(7):1648-1656
6 LUO Zhi-Gang,HE Jiu-Ming,LIU Yue-Ying,LI Tie-Gang,HE Jing-Jing,ZHANG Si-Chun,ZHANG Xin-Rong,ABLIZ Zeper.Sci.Chin.Chem.B,2014,44(5):795-800罗志刚,贺玖明,刘月英,李铁刚,何菁菁,张四纯,张新荣,再帕尔·阿不力孜.中国科学:化学,2014,44(5):795-800
7 Cornett D S,Reyzer M L,Chaurand P,Caprioli R M.Nat.Methods,2007,4(10):828-833
8 PEI Xing-Li,HUANG Yu-Yu,GONG Can,XU Xu.Chinese J.Anal.Chem.,2017,45(8):1155-1164裴兴丽,黄煜宇,龚灿,许旭.分析化学,2017,45(8):1155-1164
9 Takáts Z,Wiseman J M,Gologan B,Cooks R G.Science,2004,306:471-473
10 Wu C,Dill A L,Eberlin L S,Cooks R G,Ifa D R.Mass Spectrum.Rev.,2013,32(3):218-243
11 Liu H,Li W,He Q,Xue J,Wang J,Xiong C,Pu X,Nie Z.Sci.Rep.,2017,7:41954
12 Dill A L,Eberlin L S,Zheng C,Costa A B,Ifa D R,Cheng L,Masterson T A,Koch M O,Vitek O,Cooks R G.Anal.Bioanal.Chem.,2010,398(7-8):2969-2978
13 Luo Z,He J,Chen Y,He J,Gong T,Tang F,Wang X,Zhang R,Huang L,Zhang L,Lv H,Ma S,Fu Z,Chen X,Yu S,Abliz Z.Anal.Chem.,2013,85(5):2977-2982
14 He J,Luo Z,Huang L,He J,Chen Y,Rong X,Jia S,Tang F,Wang X,Zhang R,Zhang J,Shi J,Abliz Z,Anal.Chem.,2015,87(10):5372-5379
15 Song X,Luo Z,Li X,Li T,Wang Z,Sun C,Huang L,Xie P,Liu X,He J,Abliz Z.Anal.Chem.,2017,89(12):6318-6323
16 Li T,He J,Mao X,Bi Y,Luo Z,Guo C,Tang F,Xu X,Wang X,Wang M,Chen J,Abliz Z.Sci.Rep.,2015,5:14089
17 Corbin K D,Zeisel H S.Curr.Opin.Gastroenterol.,2012,28(2):159-165
18 Guder W G,Ross B D.Kidney Int.,1984,26(2):101-111
19 Bagnasco S,Balaban R,Fales H M,Yang Y M,Burg M.J.Biol.Chem.,1986,261(13):5872-5877
20 Sizeland P C,Chambers S T,Lever M,Bason L M,Robson R A.Kidney Int.,1993,43(2):448-453
21 Liu Y,Yan S,Ji C,Dai W,Hu W,Zhang W,Mei C.Kidney Blood Press Res.,2012,35(5):373-381
22 Eder L,Hirt L,Dunant Y.Nature,1976,264(5582):186-188
23 Karachalias N,Babaei-Jadidi R,Rabbani N,Thornalley P J.Diabetologia,2010,53(7):1506-1516
24 di Paolo G,de Camilli P,Nature,2006,443(7112):651-657
25 WANG Zhong-Hua,CHEN Yan-Hua,XU Jing,CHEN Huili,ZHOU Xia,XIAO Xin-Hua,PING Fan,HE Jiu-Ming,ABLIZ Zeper,ZHANG Rui-Ping.Chinese J.Anal.Chem.,2017,45(5):674-680王中华,陈艳华,徐婧,陈蕙莉,周霞,肖新华,平凡,贺玖明,再帕尔·阿不力孜,张瑞萍.分析化学,2017,45(5):674-680
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