An investigation on the ion/molecule reactions in SiCl4 provides critical insights into the plasma chemistry of silicon-based materials. In this study, ion/molecule reactions in a gas mixture of SiCl4/N2 were investigated using a pulsed-electron beam mass spectrometer. The primary reaction products from the reactions of N2 + and N4 + with SiCl4 were found to be SiCl3 + and SiCl4 +, respectively. The thermochemical stabilities of SiCl3 +(SiCl4)1,2, SiCl3 +(N2), and SiCl4 +(SiCl4)1,2 were determined. The discontinuous decrease in the bond energies of SiCl3 +(SiCl4) n and SiCl4 +(SiCl4) n between n = 1 and 2 indicates the participation of a covalent bond (i.e., charge transfer) in SiCl3 +(SiCl4) and SiCl4 +(SiCl4). In contrast, the bond in SiCl3 +(N2) was found to be mainly electrostatic.
Native mass spectrometry (MS) enables the direct observation of non-covalent protein interactions while preserving the higher-order structures of protein complexes. We previously developed direct native MS approaches for analyzing overexpressed protein complexes from crude lysates and single human cells, retaining their interactions with small molecules and drugs. Here, we demonstrate that this strategy can be extended to endogenous protein complexes, which are present at substantially lower abundance than the overexpressed protein complexes analyzed in previous direct native MS studies, by directly analyzing crude lysates of cultured human HEK293T cells without prior purification or buffer exchange. Although crude human cell lysates contain salts and matrix compounds that typically cause peak broadening and signal suppression, we successfully observed the endogenous ~145-kDa glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tetramer directly from unpurified human cell lysates, including its NAD+-bound forms. These results demonstrate the feasibility of directly detecting an endogenous high-molecular-weight protein complex and its cofactor-binding states from crude human cultured cell lysates, providing a basis for extending this approach to other endogenous protein assemblies and future single-cell native MS analyses.
The precise control of specific protein expression in target cells remains a critical challenge for the advancement of messenger RNA (mRNA) therapeutics. While incorporating regulatory sequences, such as microRNA-responsive RNA switches, is a promising approach for cell-type-specific targeting, optimizing these sequences requires extensive and time-consuming screening using conventional evaluation methods. In this study, we applied a multiplexed evaluation methodology utilizing peptide barcodes coupled with liquid chromatography-mass spectrometry (LC-MS) to improve the screening and design efficiency of mRNA expression control sequences. We designed mRNAs equipped with cell-specific expression control sequences and fused unique peptide barcodes to the encoded target proteins. By comparing conventional individual transfections with simultaneous co-transfections, we demonstrated that our LC-MS-based method can successfully distinguish and quantify protein expression derived from individual mRNA candidates within a single mixed sample. The simultaneous evaluation using peptide barcodes yielded results equivalent to the individual evaluations but required a reduced number of samples. This multiplexed technology would accelerate the screening process for regulatory sequences and is expected to enhance the development of mRNA therapeutics with high targeting specificity.
