方法文章

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

DOI：

10.3791/59875

⸱

2019年6月21日

 ,  ,  ,  , 
1Department of Chemistry and Biochemistry, University of Oklahoma

本文内容

摘要

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开发了一个集成的细胞操作平台,用于与单探针质谱设置结合使用,用于在环境条件下对单个悬浮单元进行在线分析。

摘要

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单细胞质谱法(SCMS)能够对单个细胞水平上广泛的细胞物种进行灵敏的检测和准确分析。单探头,一个微尺度采样和电光装置,可以配合质谱仪在线,快速SCMS分析细胞成分的环境条件。以前,单探针SCMS技术主要用于测量固定在基板上的细胞,限制用于研究的细胞类型。在目前的研究中,单探针SCMS技术已与细胞操作系统集成,通常用于体外受精。此集成的细胞操作和分析平台使用细胞选择探针捕获已识别的单个浮细胞,并将细胞转移到微尺度分解的单探针尖端,然后立即进行质谱分析。这种捕获和转移过程在分析之前从周围的溶液中去除细胞,最大限度地减少了质谱分析中基质分子的引入。这种集成设置能够分析体液样本(如尿液、血液、唾液等)中存在的靶向患者分离细胞的SCMS分析,从而允许SCMS分析在人类医学和疾病生物学中的潜在应用。

引言

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人类生物学,特别是疾病生物学,越来越被理解为单个细胞水平上活动的结果,但传统的分析方法,如液相色谱质谱法(LCMS),通常被用来分析从细胞群制备的样本,而获得的分子信息不能准确表示单个细胞层面的化学过程。这些标准,传统方法无法辨别细胞异质性对分析测量的影响,以及破坏和混合细胞制备解热剂的过程可能导致细胞的改变或损失组件1,2.传统方法的这些局限性在分析患者细胞时尤为重要,在分析患者细胞时,获得的样本可以包含许多不同细胞类型的复杂混合物。为了克服这些缺陷,单细胞分子分析方法,包括单细胞质谱法(SCMS)方法,正在越来越多地被开发并应用于生物分析,特别是细胞代谢物和低分子量生物分子3,4。

第一批SCMS技术开发使用真空技术,在非环境条件下执行分析2,5,6,7,8,9, 10,11.非环境SCMS技术能够分析细胞脂质和代谢物,但在人工条件下需要样品预处理,因此不适合实时分析。非环境分析的样品制备过程包括添加基质成分,这种制备可以改变细胞成分从自然环境12。因此,环境质谱 (MS) 技术(对于采样环境不需要真空)用于分析接近原生环境中的细胞。没有真空环境,实验设计具有多功能性;可以添加摄像头来监控细胞过程,软电离技术可以与分离技术相结合,从每个单细胞实验4、12、13中接收更好的信息。 ,14,15,16,17,18,19,20,21,22 ,23,24,25,26,27,28,29,30,31 ,32,33,34,35,36,37,38,39,40 ,41,42.

单探针SCMS方法是一种环境技术,分析近原生环境中的活哺乳动物癌细胞系21,43,44,45,46。此外,单探针装置还用于其他质谱学应用,包括多细胞球体细胞外分子分析以及组织47、48、49的MS成像 ,50,51,52.然而,由于这种方法需要在基质上进行细胞固定,因此不能直接使用这种技术3,53分析悬浮细胞。因此,单探针SCMS系统不能直接用于样品非粘附单细胞,如从患者血液或其他体液中分离的非粘附细胞系或悬浮细胞54。在这项工作中,集成细胞操作平台(ICMP)与单探针SCMS技术相结合,以最少的样品制备在线分析活的悬浮细胞(图1)46。ICMP 包括一个倒置显微镜来监测细胞选择,一个玻璃细胞选择探头,一个用于捕获单个浮动细胞的显微注射器,一个用于保持细胞温度的加热板,两个细胞操作系统来控制空间玻璃细胞选择探针和单探针的运动,以及数字显微镜的运动,以观察细胞从细胞选择探针尖端到单探针尖端的细胞转移。单探的制作在以前的出版物中已详细说明,不会在此讨论21,48。ICMP/单探头系统与高分辨率质谱仪耦合。这种集成设置允许从复杂的生物样本中取样识别的单个细胞,而基质分子的影响最小。

方案

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1. 玻璃电池选择探头制造

  1. 将单孔玻璃管转换为带尖尖的锥形探头。
    1. 将单孔玻璃管(ID:0.3 mm,OD: 1.1.mm)放入垂直移液器支架的夹子中,将玻璃相对于加热线圈居中并拧紧以将管固定到位。加热线圈由 18 度耐镍铬导线(长度约 60 mm)组成,缠绕在金属棒(直径 = 3.90 mm)周围 2.5 倍。
    2. 设置带温度程序 19.5(制造商装置)的玻璃管。此参数可以针对特定仪器进行修改。
    3. 将电磁阀柱塞设置为 4(制造商装置)。此参数可以针对特定仪器进行修改。
    4. 触发电磁阀以拉出玻璃管。此步骤创建两个在尖端融合的探头。
    5. 使用钳子将 ±1 mm 切离每个探头的尖端 1 毫米,在探头尖端处创建直径为 ±10 μm 的孔。
  2. 弯曲玻璃探头,以便轻松耦合到 ICMP/单探头 SCMS 设置。
    1. 将拉玻璃探头插入微锻件中,将尖端 +3 mm 放置在铂金加热导线上方。
    2. 将铂线的热量调低至最高温度的 30%。
    3. 从原始位置弯曲探头 [45](图 2)。

2. 集成细胞操作平台组装

  1. 将倒置显微镜、显微注射器和两个细胞操作系统放在电动表上,以便与质谱仪轻松耦合。
    1. 修改其中一个单元操作系统,通过用臂夹替换端部来容纳单探头。
    2. 使用带针的塑料注射器将矿物油填充到显微注射器中。避免油管中的气泡,因为这将影响吸力。
    3. 将倒置显微镜的台式插入件更换为加热板。在分析之前,将加热板设置为 37°C。
  2. 设置玻璃单元选择设备。
    1. 将长(非弯曲)侧插入毛细管支架,并拧紧螺钉以将探头固定到位,将玻璃电池选择探头插入显微喷油器的金属支架内。将探头尖端的角度与加热板平行放置。
      注意:玻璃探头非常锋利和脆弱,很容易断裂。将探头插入显微注射器时,请保护眼睛并格外小心。
    2. 将显微注射器的金属支架固定到细胞操作系统中。将探头尖端靠近倒置显微镜灯的中间。

3. 为质谱仪入口创建扩展的电传递管

  1. 使用金属切割机切割一块不锈钢管(OD:0.0625 (1/16),ID: 0.021 英寸) ±250 mm 的长度。
  2. 测量135毫米从末端,并放置金属铁,使+135毫米将暴露在大气中和+115毫米将在质谱仪内。用两个扳手固定铁圈以将其拧紧。

4. 将 ICMP 与单探针设置耦合

  1. 将包含单探头的玻璃滑块固定到细胞操作系统的臂夹中。
    注:单探针是根据先前公布的协议48制造的,当前研究中有两个小变化:纳米ESI发射体更长,便于与质谱仪耦合,单探针粘附在玻璃上右侧,以避免干扰玻璃细胞选择装置的空间运动(图2)。
  2. 通过将毛细管放入塑料套管的套筒(1/16 x .005 in)中,并用手指拧紧接头,将提供溶剂的毛细管连接到导电接头。
    1. 通过将毛细管放入套管(1/32 x .007)并拧紧接头,将导电接头的另一侧连接到毛细管(ID:40 μm,OD: 150 μm),该毛细管连接到装有取样溶剂的注射器。在这些实验中,使用含0.1%甲酸的醋酸作为取样溶剂。
      注:取样溶剂是灵活的,但它应主要含有乙酰乙酰乙酰烯(或乙酰乙酰乙酸甲酸,以更好地电离),用于快速微尺度细胞溶解。
    2. 将注射器固定到质谱仪上的注射器泵中。
    3. 将电电化电压线放在连接到导电结合的铜线上。
  3. 将纳米 ESI 发射器 ±1 mm 定位到扩展的输水管的孔。
    1. 使用细胞操作系统控制单探针的空间运动,并将纳米ESI发射器集中放置在扩展的输油管前面。

5. 悬浮细胞样品制备

  1. 分析前一天(+18-24小时),在细胞培养瓶(T25)中播种细胞进行测试。K562人骨髓性白血病细胞作为模型在这项研究中使用。
    1. 加热1x磷酸盐缓冲盐水(PBS)和罗斯威尔公园纪念研究所(RPMI)介质辅以10%合成胎儿牛血清(FBS)和1%青霉素-链霉素在37°C30分钟。
    2. 种子 #1 x 106细胞,总体积为 10 mL,将细胞与热培养基结合。通常,使用 10 mL 移液器将 8 mL 的 RPMI 介质放入细胞培养瓶中。然后,使用 2 mL 移液器将 2 mL 的汇入 K562 细胞培养基用于 ±1 x 106细胞。
    3. 在37°C和5%CO2下孵育细胞,直到分析。
  2. 准备单元格进行分析。
    1. 移液细胞从细胞培养瓶到15mL离心管。
    2. 在400 x g和37°C下旋转细胞5分钟,并丢弃上清液。
    3. 在4 mL的RPMI培养基中重新悬浮细胞,在所需的治疗浓度下含有药物化合物。
      注:对于控制单元的分析,在RPMI培养基的4mL中重新悬浮细胞,并跳到步骤6。
    4. 在37°C和5%CO2的疗程中孵育细胞。
    5. 在400 x g和37°C下旋转细胞5分钟。吸出上清液。
    6. 细胞在10 mL的PBS中重新悬浮,在400 x g和37°C下离心5分钟。旋转后,丢弃上清液。重复此步骤 3 次,以尽量减少从细胞外成分中检测药物。
    7. 在4 mL的PBS中重新悬浮细胞进行分析。

6. 使用 ICMP/单探头设置执行 SCMS 测量

  1. 为实验定制质谱仪的参数。
    1. 在仪器软件的"扫描模式"标题下,选择"定义扫描"。在m/z 400、1 微扫描、100 ms 最大喷射时间下使用 60,000 m/m 的分辨率,并打开自动增益控制 (AGC)。实验使用了100-1000的质量范围(m/z)。参数可以根据仪器型号进行修改。
    2. 在注射器泵下,选择150 nL/min的流速,需要针对每个实验优化流量。
    3. 选择NSI 电源并施加 ±4.5 kV 的电压。此参数还需要针对每个实验进行优化。
  2. 打开倒置显微镜(为顶板和底透镜选择 40 倍放大倍率),并将其连接到笔记本电脑的 USB 端口,以捕获实时视频源。打开加热板并将其设置为 37°C。
  3. 在计算机上,转到"获取数据"选项卡,并在"获取时间"下选择"连续"。
  4. 准备样品进行分析。
    1. 将 2-3 mL 的样品放入小培养皿(35 mm x 12 mm)的盖子中。
    2. 6.4.2 将样品从倒置显微镜置于加热板顶部的光线中心。
  5. 准备玻璃电池选择探头进行分析。使用细胞操作系统移动探头,使其尖端与细胞位于同一平面的倒置显微镜下。
  6. 选择要分析的单个单元格。
    1. 使用单元格操作系统将单元格选择探针尖端移动到目标单元格。使用倒置显微镜监控此过程。
      注: 如果细胞选择探头的尖端不能与细胞位于同一平面上,则探头的弯曲部分可能未适当倾斜。调整细胞选择探针的位置,直到两个探针尖端可以与显微镜下的细胞一起聚焦。
    2. 轻轻转动显微注射器手柄,以调整油管内矿物油的位置。微注射器提供温和的吸力,将目标细胞固定到细胞选择探针尖端。
      注:如果细胞选择探头无法通过吸力捕获细胞,请检查细胞选择探头以确保其完全插入毛细管支架。此外,检查微注射器和油管中的矿物油含量,并排出空气(如果有)。
    3. 使用细胞操作系统将细胞选择探针尖端处的细胞移动到单探针尖端,使用聚焦在单探针尖端的数字显微镜来监控此过程。接触时,单探针尖端处的小醋酸滴会迅速使细胞发生分管,然后细胞流化立即进行离子化,进行在线MS分析。
      注: 由于所选电池通过温和的吸力固定在细胞选择探针尖端上,因此在转移到单探针尖端期间,该电池可能会分离。因此,如果在 5 秒内未观察到典型细胞脂质的 ion 信号(参见下面的代表性结果),则细胞可能变得未连接,并且需要选择不同的细胞。

结果

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首先,采用未经处理的K562细胞建立实验方法。在典型的SCMS实验中,通过转移细胞、检测细胞内容物和完成测量后,可以观察到质谱的明显变化(图S1)。监测三种常见的细胞脂质峰(磷脂脂,PC),包括PC(34:4)(m/z 754.536)、PC(36:4)(m/z 782.567)和PC(38:5)(m/z 808.583),以确保细胞成功转移和细胞检出含量(图S2)二十一、四十三、四十六、五十五、五十六。如果在 5 秒内看不到脂质峰值,则微注射器中的矿物油位将改变,以减少在细胞选择探针尖端保持电池的吸力;需要谨慎,以便不会将矿物油从细胞选择探头中推出。在未处理的细胞解致样品上,使用MS/MS确认许多PC在m/z 750-850质量范围内的身份(图3,图S2,表1)46。

K562细胞也接受各种药物化合物的治疗,以扩大该方法的多功能性。K562细胞分别孵育4小时和2小时,分别使用Gemcitabine(1 μM)和分醇(1μM)孵育1小时和OSW-1(100 nM,1 μM)。然后用PBS清洗细胞,以尽量减少从细胞外含量中检测药物化合物。基质(例如,细胞培养基、PBS和溶剂中的离子)对细胞内容物质谱的贡献可以通过数据减法消除,因为它们离子信号明显不同(图S3)。所有三种药物化合物均使用ICMP/单探针MS设置(图S4)46检测。这些结果表明,该方法可用于研究近原生环境中溶液中细胞的单细胞水平上的细胞内脂质、药物和代谢物。

figure-results-1
图 1.单悬架单元MS实验的实验设置。(A) 集成细胞操作平台 (ICMP) 与质谱仪相结合。(B) 用于分析悬浮细胞的原理图.(C) 使用细胞选择探针选择的 K562 细胞的实验视图。经Standke等人许可转载。版权所有 2019 美国化学学会。请点击此处查看此图的较大版本。

figure-results-2
图 2.用于单悬架细胞MS实验的经过修改的单探针和细胞选择探头的照片。请点击此处查看此图的较大版本。

figure-results-3
图 3.从单个细胞放大质量谱,显示代表性物种(m/z 750-850)。使用 MS/MS 分析 (图 S1) 确认化学结构。转载经斯坦克等人46许可。版权所有 2019 美国化学学会。请点击此处查看此图的较大版本。

药物分子|m/z质量误差(ppm)
[宝石 ] H]+ 264.07611.32
[税务 + 纳]+ 876.3182.74
[OSW-1 = Na]+ 895.4450.89
细胞脂质
[PC (34:4) + H]+ 754.5353.71
[PC (34:3) + H]+ 756.5513.44
[PC (34:2) + H]+ 758.5690.66
[PC (36:5) + H]+ 780.5513.07
[PC (36:4) + H]+ 782.5682.17
[PC (36:3) + H]+ 784.5850.64
[PC (38:7) + H]+ 804.5514.1
[PC (38:6) + H]+ 806.5672.48
[PC (38:5) + H]+ 808.5832.72
[PC (38:4) + H]+ 810.6010
[PC (40:7) + H]+ 832.5833.12

表 1.使用 ICMP/单探针设置识别蜂窝组件。通过比较MS/MS结果和标准化合物,证实了所有药物化合物的检测。

讨论

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集成的细胞操作和分析平台旨在扩展单探针MS方法的多功能性,允许在接近原生环境中对非粘附细胞进行在线、快速分析。该技术的一个主要优点是,需要最少的样品制备,因此在模拟其标准状态的条件下分析细胞。特别是,可以直观地识别和选择感兴趣的单个细胞,将基质效应对MS电化效率的影响降至最低,同时保持细胞在自然环境中,因此其结果是更具代表性的细胞的原生状态 (图 S3)。该技术可能用于研究悬浮在生物液体中的患者细胞,该技术的另一个优点是灵活选择取样溶剂。将醋酸酯作为主要取样溶剂非常重要,以便迅速发生微尺度溶解。内部标准(例如,异位标记药物化合物)可能添加到取样溶剂中,用于定量单个细胞的感兴趣的分子(例如药物分子),包括那些可在革命中发挥关键作用的分子。个性化药物治疗在未来54。

虽然这个集成系统可以很方便地用于分析广泛的细胞范围,但该方法的一个局限性是,单探针和单元选择探头都不具有商业用途;在每次实验之前,需要优化许多参数(例如,流速、电压、纳米ESI发射器和电传递管之间的长度等)。此外,由于单探针和细胞选择探头的体积小,环境扰动(例如气流)可能会导致难以在两个探头之间建立连接。短期解决方案是将细胞选择探头弯曲到接近末端,以尽量减少锥形的长度。今后的工作包括开发一个住房,以包围设置的关键部分,以尽量减少对环境的影响。由于细胞含量有限,从细胞获取时间短(±2-3s),MS/MS分析只能对相对丰富的物种进行。影响检测灵敏度的其他因素包括由于引入基质以及细胞和通过扩展的电传递管而潜在的电因损失而抑制的电化效率。

披露

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

作者没有什么可透露的。

致谢

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

作者感谢那加拉马·科塔帕利在为悬浮细胞和细胞莱沙实验开发样品制备方面所做的工作。此外,作者感谢NIH(R01GM116116和R21CA204706)的资助。

材料

本文使用的材料清单
姓名公司目录编号评论
乙腈Millipore Co.AX0145-1采样溶剂
CellTram VarioEppendorf6221ICMP
铜线stores.ebay.com/jewelerheaven死软，圆形，20 号，25 英尺导电接头装置
数字立体显微镜深圳 D&F 公司Supereyes T004分析
一次性微量移液器，1-5 和微量;LRochester Scientific5065细胞选择探针制造
双孔石英管，1.120"x0.005"x12"Friedrich &Dimmock 公司MBT-005-020-2Q单探针制造
环氧树脂Devcon零件编号 20945单探针制造
Eppendorf 细胞作系统EppendorfTransferman NK517800397-U.R.国际计量局
外部螺母VALCO*CHEMINERTEN1离子传输管制造
甲酸Sigma-Aldrich399388-500ML采样溶剂
熔融石英毛细管，ID：40 µm，外径：100 µmPolymicro TechnologiesTSP040105单探针制造，导电接头设置
熔融石英毛细管，ID：50 µm，外径：150 µmPolymicro Technologies1068150015导电接头装置
HyClone 合成胎牛血清 （FBS）Fischer SciSH3006603细胞培养
在线 MicroFilterIDEX Health &Science LLCM-520导电接头装置
激光拉拔器Sutter Instrument Co.型号 P-2000单探针制造
LED 紫外灯佛山亮雅牙科设备LY-C240单探针制造
LTQ Orbitrap 质谱仪Thermo ScientificLTQ Orbitrap XL分析
MicroforgeNarishige， Co.MF-9细胞选择探针制造
MicrounionIDEX Health &Science LLCM-539导电接头
PEEK 管，1/32x0.005x 5 英尺IDEX Health &Science LLC1576导电接头设置
PEEK 管，1/32x0.007x 5 英尺IDEX Health &Science LLC1577导电联合装置
青霉素/链霉素Gibco/Life Technologies15140-122细胞培养
培养皿，35x10 mmVWR25382-334样品制备
磷酸盐缓冲盐水 （PBS）VWR0780-50L细胞培养
铂丝Narishige， Co.型号 PT-AMicroforge
电源尼康PSM-2120ICMP
RPMI，1X 带康宁 glutagro康宁10-104-CV细胞培养
单孔管Boralex5065细胞选择探针制造
不锈钢密封垫圈，用于 1/16" ODIDEX Health 和Science LLCVHP-200-01x离子转移管制造
不锈钢管，1/32x 205 µm x30 cmIDEX 健康 &Science LLCU-1128离子转移管制造
注射器，250 µLHamilton1725LTN250UL进样注射器
T25 培养瓶CellStar690160细胞培养
Thermo LTQ XL 离子源接口法兰新物镜PB5500分析
ThermoPlateTokaiHit55R30NICMP
TrypLE ExpressGibco12605-010细胞培养
切管器，用于 1/16"不锈钢SUPELCO58692-U离子传输管制造
USB 数码摄影显微镜dx.comSO2 25~500X分析
UV 固化树脂Prime Dental货号 006.030单探针制造
立式移液器拉拔器David Kopf Instruments720型 电解池选择探针制造
电压外壳PicoChipPCH-A00120ICMP/MS 接口
剪线Craftsman4 1/2 英寸末端钳导电接头设置
钳

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标签

Single Cell Mass SpectrometryCell Manipulation SystemSingle Probe SCMSSuspension Cell AnalysisMass Spectrometry AnalysisIntracellular Metabolite DetectionNanoelectrospray IonizationPatient Derived CellsDrug QuantificationCellular Heterogeneity

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