Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
Division 1.1 - Inorganic Trace Analysis, Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Str. 11, 12489 Berlin, Germany.
Anal Chem. 2023 Sep 5;95(35):13322-13329. doi: 10.1021/acs.analchem.3c02558. Epub 2023 Aug 11.
An automated and straightforward detection and data treatment strategy for the determination of the protein relative concentration in individual human cells by single cell-inductively coupled plasma-time-of-flight mass spectrometry (sc-ICP-ToF-MS) is proposed. Metal nanocluster (NC)-labeled specific antibodies for the target proteins were employed, and ruthenium red (RR) staining, which binds to the cells surface, was used to determine the number of cell events as well as to evaluate the relative volume of the cells. As a proof of concept, the expression of hepcidin, metallothionein-2, and ferroportin employing specific antibodies labeled with IrNCs, PtNCs, and AuNCs, respectively, was investigated by sc-ICP-ToF-MS in human ARPE-19 cells. Taking into account that ARPE-19 cells are spherical in suspension and RR binds to the surface of the cells, the Ru intensity was related to the cell volume (i.e., the cell volume is directly proportional to (Ru intensity)), making it possible to determine not only the mass of the target proteins in each individual cell but also the relative concentration. The proposed approach is of particular interest in comparing cell cultures subjected to different supplementations. ARPE-19 cell cultures under two stress conditions were compared: a hyperglycemic model and an oxidative stress model. The comparison of the control with treated cells shows not only the mass of analyzed species but also the relative changes in the cell volume and concentration of target proteins, clearly allowing the identification of subpopulations under the respective treatment.
提出了一种自动化、简便的检测和数据处理策略，用于通过单细胞电感耦合等离子体质谱飞行时间质谱法（sc-ICP-ToF-MS）测定单个人类细胞中蛋白质的相对浓度。使用针对靶蛋白的金属纳米团簇（NC）标记的特异性抗体，并用钌红（RR）染色结合细胞表面，以确定细胞事件的数量，并评估细胞的相对体积。作为概念验证，使用分别用 IrNCs、PtNCs 和 AuNCs 标记的特异性抗体，用人 ARPE-19 细胞研究了铁调素、金属硫蛋白-2 和亚铁蛋白的表达，通过 sc-ICP-ToF-MS。考虑到 ARPE-19 细胞在悬浮液中呈球形，并且 RR 与细胞表面结合，因此 Ru 强度与细胞体积（即细胞体积与 Ru 强度成正比）相关，从而不仅可以确定每个单个细胞中的靶蛋白质量，还可以确定相对浓度。该方法特别适用于比较接受不同补充的细胞培养物。比较了两种应激条件下的 ARPE-19 细胞培养物：高血糖模型和氧化应激模型。与处理过的细胞相比，对照细胞的比较不仅显示了分析物的质量，还显示了细胞体积和靶蛋白浓度的相对变化，这显然允许在各自的处理下识别亚群。
