质谱成像｜原位酶解蛋白质谱成像中细胞水平空间分辨率的蛋白鉴定方法建立

原位酶解已成为质谱成像(MSI)鉴定蛋白质的首选方法。德国的拜罗伊特大学Andreas Römpp课题组报告了组织原位酶解后质谱成像的数据采集和蛋白质鉴定方面的进展。本研究在空间分辨率、蛋白质鉴定和数据处理方面的结果对组织上原位酶解MSI领域的发展意义重大。

作者在50 μm空间分辨率的小鼠脑冠状切面上检测酶解多肽，m/z=200处的质量分辨率为140,000，图一展示了髓磷脂碱性蛋白的酶解多肽HGFLPR + H⁺ (m/z =726.40456 ± 0.005) 在胼胝体和尾壳核区域的高分布，这与髓鞘碱性蛋白主要位于脑白质作为髓鞘的一部分吻合。该结果展示了精细的室管膜结构(由一到两层细胞组成)，并H&E染色在组织学方面验证该结果。这表明，以细胞分辨率或接近细胞分辨率的质谱成像是可以实现的。

Fig. 1 MSI of tryptic peptides in a coronal mouse brain section, imaged with 50 μm pixel size (85 × 135 pixels) and a mass resolution of 140,000 (at m/z = 200). a Overlay of selected ion images of tryptic peptide GTGASGSFK + Na⁺ (green) corresponding to histone 1 protein (P15864), tryptic peptide HGFLPR + H⁺ (blue) corresponding to myelin basic protein (Q09J72), and tryptic peptide DLSAGAVSAVR + H⁺ (red) corresponding to the protein isoaspartyl peptidase/l-asparaginase (Q8C0M9). b Magnification of the ependyma area, H&E stained. C Mass spectrum of a single pixel (50 μm pixel size) of tryptic peptide GTGASGSFK + Na⁺ corresponding to histone 1 protein (P15864) which is located in ependyma, consisting of one to two cell layers. d Additional tryptic peptides for P15864.

作者描述了一个详细的鉴定流程（图二），通过比较LC-MS/MS结果和原位酶解的数据，鉴定了99个蛋白质(435个对应的肽段)。这些结果是在严格的参数下获得的，包括在成像模式下的高质量准确性(RSME < 3 ppm)和每个蛋白至少有两个独特的肽段显示一致的空间分布。作者鉴定出近50%的蛋白质至少有4个相应的肽段。

Fig. 2 Workflow for tryptic peptide identification in MALDI MS imaging experiments with example numbers of the measurement shown in Fig. 1 (coronal mouse brain section with 50 μm pixel size). Part I consists of LC-MS/MS measurements to identify proteins that are present in the analyzed tissue. Parts II and III have the purpose to select suitable candidate proteins for in silico digestion. The identification of proteins in part IV is independent and can also be performed with alternative protein candidates. In this example, about 50% of proteins were identified with at least four corresponding peptides.

文章中样品制备方案也可以用于更高的空间分辨率。图三显示了该方法25 μm空间分辨率(180 × 250像素)的小鼠大脑冠状切面髓磷脂碱性蛋白的酶解多肽HGFLPR + H⁺ (m/z =726.40456 ± 0.005)的测试结果，质量分辨率为35,000 (m/z = 200)。

Fig. 3 MS imaging of coronal mouse brain section. a Myelin staining of adjacent tissue section. b Selected ion image with a pixel size of 25 μm of tryptic peptide HGFLPR + H⁺ (m/z = 726.40456 ± 0.005) corresponding to myelin basic protein.

图四展示了在25 μm空间分辨率的小鼠大脑的不同区域的测试结果，小脑区采用50 × 65像素，质量分辨率R = 70,000 (m/z = 200)。成像结果显示微管蛋白聚合促进蛋白的原位酶解多肽AKPAK + Na⁺分布与颗粒层的结构相关，同时H&E染色在组织学方面验证该结果。

Fig. 4 Influence of spatial resolution and imaging accuracy in a measurement of mouse brain cerebellum with 25 μm pixel size shown on m/z = 536.316703 (tryptic peptide of tubulin polymerization-promoting protein). a–c selected ion images recalculated for different acquisition parameters. d Selected ion image measured with 25 μm pixel size and generated with a small bin width of Δm/z = ± 0.005 U. e ALLEN mouse brain atlas: in situ hybridization of tubulin polymerization-promoting protein. f H&E staining of an adjacent section.

初期的工作集中在分析作为模型系统的小鼠脑组织。然而，酶解条件和数据采集在很大程度上取决于组织的性质。因此，作者对该实验方案应用不同的样品类型进行了评估。如图五所示，用50 μm (720 ×330像素)的空间分辨率和质量分辨率为70,000（m/z= 200）对6日龄小鼠的全身切片进行测试分析。多种可能的酶解多肽被检测到，它们位于各个器官，并在切片成像和H&E染色中显示出良好的图像相关性。

Fig. 5 Whole mouse body section. a Photograph of block-face embedded in CMC during cutting process. b MALDI MS image: overlay of selected ion images of m/z = 1045.56365 ± 0.005 (blue), m/z = 519.32492 ± 0.005 (green), m/z = 530.25862 ± 0.005 (yellow), and m/z = 612.28662 ± 0.005 (yellow). c H&E-stained section after measurement.

在大中华区独家代理的两款质谱成像离子源，都可搭载Thermo Scientific^TM Q Exactive^TM或Obitrap Exploris^TM系列质谱仪。

AP-SMALDI 5AF高分辨自动聚焦3D快速质谱成像系统，常压操作环境，空间分辨率可达到3 μm，独特3D检测模式可以检测凹凸不平的样品表面，快速检测模式可达18pixel/s，全像素检测大大提高检测灵敏度，高空间分辨率和高质量分辨率使样本中的分子化合物达到最佳成像效果。

MALDI ESI Injector^TM 透射式超高分辨质谱成像系统，可以同时搭载MALDI离子源与ESI离子源，既可用于传统LC-MS/MS实验，也可用于质谱成像检测，通过双离子漏斗接口实现离子源快速切换，无需拆卸，操作便捷，并且接口可以进一步升级为MALDI-2和t-MALDI检测，大大提高空间分辨率和检测灵敏度。