The gas-phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o-TEMPO-Bz-conjugated peptides with an intra- and intermolecular disulfide bond was investigated using MS(n) tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLCRIVVIRVCR), TGF-α (CHSGYVGVRC), MCH (DFDMLRCMLGRVFRPCWQY) and Adrenomedullin (16-31) (CRFGTCTVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o-TEMPO-Bz-conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the S-S or C-S bond was readily cleaved. The observed peptide backbone fragments included a-, c-, x- or z-types, which indicates that the radical-driven peptide fragmentation mechanism plays an important role in TEMPO-FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the S-S or C-S bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of •SH or •SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the S-S bond, the abstraction of a hydrogen atom at C(β) by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H-abstraction at C(α) is suggested to lead to C-S bond cleavage, which yields [ion ± S] fragments or the loss of •SH or •SSH.

Although tetraarsenic hexoxide is known to exert an anti-tumor effect by inducing apoptosis in various cancer cells, its effect on other forms of regulated cell death remains unclear. Here, we show that tetraarsenic hexoxide induces the pyroptotic cell death through activation of mitochondrial reactive oxygen species (ROS)-mediated caspase-3/gasdermin E (GSDME) pathway, thereby suppressing tumor growth and metastasis of triple-negative breast cancer (TNBC) cells. Interestingly, tetraarsenic hexoxide-treated TNBC cells exhibited specific pyroptotic characteristics, including cell swelling, balloon-like bubbling, and LDH releases through pore formation in the plasma membrane, eventually suppressing tumor formation and lung metastasis of TNBC cells. Mechanistically, tetraarsenic hexoxide markedly enhanced the production of mitochondrial ROS by inhibiting phosphorylation of mitochondrial STAT3, subsequently inducing caspase-3-dependent cleavage of GSDME, which consequently promoted pyroptotic cell death in TNBC cells. Collectively, our findings highlight tetraarsenic hexoxide-induced pyroptosis as a new therapeutic strategy that may inhibit cancer progression of TNBC cells.

Peptide dissociation behavior in TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-based FRIPS (free radical initiated peptide sequencing) mass spectrometry was analyzed in both positive- and negative-ion modes for a number of peptides including angiotensin II, kinetensin, glycoprotein IIb fragment (296-306), des-Pro(2)-bradykinin, and ubiquitin tryptic fragment (43-48). In the positive mode, the ·Bz-C(O)-peptide radical species was produced exclusively at the initial collisional activation of o-TEMPO-Bz-C(O)-peptides, and two consecutive applications of collisional activation were needed to observe peptide backbone fragments. In contrast, in the negative-ion mode, a single application of collisional activation to o-TEMPO-Bz-C(O)-peptides produced extensive peptide backbone fragmentations as well as ·Bz-C(O)-peptide radical species. This result indicates that the duty cycle in the TEMPO-based FRIPS mass spectrometry can be reduced by one-half in the negative-ion mode. In addition, the fragment ions observed in the negative-ion experiments were mainly of the a-, c-, x-, and z-types, indicating that radical-driven tandem mass spectrometry was mainly responsible for the TEMPO-based FRIPS even with a single application of collisional activation. Furthermore, the survival fraction analysis of o-TEMPO-Bz-C(O)-peptides was made as a function of the applied normalized collision energy (NCE). This helped us to better understand the differences in FRIPS behavior between the positive- and negative-ion modes in terms of dissociation energetics. The duty-cycle improvement made in the present study provides a cornerstone for future research aiming to achieve a single-step FRIPS in the positive-ion mode.