RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

Comparative analysis of constitutes and metabolites for traditional Chinese medicine using IDA and SWATH data acquisition modes on LC-Q-TOF MS / 药物分析学报

Identification of components and metabolites of traditional Chinese medicines (TCMs) employing liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) techniques with information-dependent acquisition (IDA) approaches is increasingly frequent. A current drawback of IDA-MS is that the complexity of a sample might prevent important compounds from being triggered in IDA settings. Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) method where the instrument deterministically fragments all precursor ions within the predefined m/z range in a systematic and unbiased fashion. Herein, the superiority of SWATH on the detection of TCMs' components was firstly investigated by comparing the detection ef-ficiency of SWATH-MS and IDA-MS data acquisition modes, and sanguisorbin extract was used as a mode TCM. After optimizing the setting parameters of SWATH, rolling collision energy (CE) and variable Q1 isolation windows were found to be more efficient for sanguisorbin identification than the fixed CE and fixed Q1 isolation window. More importantly, the qualitative efficiency of SWATH-MS on sanguisorbins was found significantly higher than that of IDA-MS data acquisition. In IDA mode, 18 kinds of sangui-sorbins were detected in sanguisorbin extract. A total of 47 sanguisorbins were detected when SWATH-MS was used under rolling CE and flexible Q1 isolation window modes. Besides, 26 metabolites of sangui-sorbins were identified in rat plasma, and their metabolic pathways could be deduced as decarbonylation, oxidization, reduction, methylation, and glucuronidation according to their fragmental ions acquired in SWATH-MS mode. Thus, SWATH-MS data acquisition could provide more comprehensive information for the component and metabolite identification for TCMs than IDA-MS.