Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA 99354, USA.
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
Lab Chip. 2022 Jul 26;22(15):2869-2877. doi: 10.1039/d2lc00384h.
Spatial proteomics holds great promise for revealing tissue heterogeneity in both physiological and pathological conditions. However, one significant limitation of most spatial proteomics workflows is the requirement of large sample amounts that blurs cell-type-specific or microstructure-specific information. In this study, we developed an improved sample preparation approach for spatial proteomics and integrated it with our previously-established laser capture microdissection (LCM) and microfluidics sample processing platform. Specifically, we developed a hanging drop (HD) method to improve the sample recovery by positioning a nanowell chip upside-down during protein extraction and tryptic digestion steps. Compared with the commonly-used sitting-drop method, the HD method keeps the tissue pixel away from the container surface, and thus improves the accessibility of the extraction/digestion buffer to the tissue sample. The HD method can increase the MS signal by 7 fold, leading to a 66% increase in the number of identified proteins. An average of 721, 1489, and 2521 proteins can be quantitatively profiled from laser-dissected 10 μm-thick mouse liver tissue pixels with areas of 0.0025, 0.01, and 0.04 mm, respectively. The improved system was further validated in the study of cell-type-specific proteomes of mouse uterine tissues.
空间蛋白质组学在揭示生理和病理条件下的组织异质性方面具有巨大的潜力。然而，大多数空间蛋白质组学工作流程的一个显著限制是需要大量的样本，这会模糊细胞类型特异性或微结构特异性信息。在本研究中，我们开发了一种改进的空间蛋白质组学样品制备方法，并将其与我们之前建立的激光捕获微切割（LCM）和微流控样品处理平台集成在一起。具体来说，我们开发了一种悬滴（HD）方法，通过在蛋白质提取和胰蛋白酶消化步骤中将纳米孔芯片倒置，来提高样品回收效率。与常用的坐滴法相比，HD 方法将组织像素与容器表面隔开，从而提高了提取/消化缓冲液对组织样品的可及性。HD 方法可以将 MS 信号增加 7 倍，从而使鉴定的蛋白质数量增加 66%。从面积分别为 0.0025、0.01 和 0.04 mm 的激光切割的 10 μm 厚小鼠肝组织像素中，可以定量分析出平均 721、1489 和 2521 种蛋白质。该改进系统在研究小鼠子宫组织的细胞类型特异性蛋白质组学方面得到了进一步验证。
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