Grgic Andrej, Cuypers Eva, Dubois Ludwig J, Ellis Shane R, Heeren Ron M A
The Maastricht MultiModal Molecular Imaging (M4I) Institute, Division of Imaging Mass Spectrometry (IMS), Maastricht University, 6229 ER Maastricht, The Netherlands.
The M-Lab, Department of Precision Medicine, GROW - Research Institute for Oncology and Reproduction, Maastricht University, 6229 ER Maastricht, The Netherlands.
J Proteome Res. 2024 Dec 6;23(12):5372-5379. doi: 10.1021/acs.jproteome.4c00528. Epub 2024 Oct 24.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) started with spatial mapping of peptides and proteins. Since then, numerous bottom-up protocols have been developed. However, achievable spatial resolution and sample preparation with many wet steps hindered the development of single cell-level workflows for bottom-up spatial proteomics. This study presents a protocol optimized for MALDI-MSI measurements of single cells within the context of their 2D culture. Sublimation of CHCA, followed by a dip in ice-cold ammonium phosphate monobasic (AmP), produced peptide-rich mass spectra while maintaining matrix crystal sizes around 400 nm. This enables MALDI-MSI imaging of proteins in single cells grown on an ITO slide with a throughput of approximately 7800 cells per day. 89 peptide-like features corresponding to a single MDA-MB-231 breast cancer cell were detected. Furthermore, by combining the MALDI-MSI data with LC-MS/MS data obtained on cell pellets, we have successfully identified 24 peptides corresponding to 17 proteins, including actin, vimentin, and transgelin-2.
基质辅助激光解吸/电离（MALDI）质谱成像（MSI）始于对肽和蛋白质的空间映射。从那时起，已经开发了许多自下而上的方案。然而，可实现的空间分辨率以及许多湿步骤的样品制备阻碍了自下而上空间蛋白质组学单细胞水平工作流程的发展。本研究提出了一种在二维培养背景下针对单细胞MALDI-MSI测量进行优化的方案。CHCA升华，然后浸入冰冷的磷酸二氢铵（AmP）中，产生了富含肽的质谱，同时将基质晶体尺寸保持在400nm左右。这使得能够对生长在ITO载玻片上的单细胞中的蛋白质进行MALDI-MSI成像，每天的通量约为7800个细胞。检测到了与单个MDA-MB-231乳腺癌细胞相对应的89个类肽特征。此外，通过将MALDI-MSI数据与在细胞沉淀上获得的LC-MS/MS数据相结合，我们成功鉴定了对应于17种蛋白质的24种肽，包括肌动蛋白、波形蛋白和原肌球蛋白-2。
