SICyLIA-cTMT enables dissecting redox proteome dynamics with high accuracy and depth at micrograms scale (PART 2 = Human mammary breast cancer associated fibroblasts with carrier channel)

Cysteine oxidative modifications are fundamental signaling events that regulate cellular functions under both physiological and pathological conditions. However, our understanding of these modifications is limited by their low abundance and the technical challenges associated with studying their dynamics in a comprehensive and unbiased manner. Here, we developed the SICyLIA-TMT workflow, which uses light and heavy iodoacetamide (IAA) to sequentially label reduced and reversibly oxidized cysteine residues within the same sample. By applying tandem mass tags (TMTs) to differentially label peptides of each experimental conditions, our strategy allows simultaneous analysis of both oxidative modification dynamics and protein levels across multiple experimental conditions from only micrograms of starting material. Furthermore, to enhance the detection of low-abundance, cysteine residues carrying oxidative modifications, we dedicated a TMT channel as carrier for heavy IAA-labelled peptides (SICyLIA-cTMT). This strategy remarkably increased the quantification of reversibly oxidized cysteine residues and enabled precise stoichiometry calculations. Last, combining SICyLIA-TMT with the latest generation of MS instruments, Orbitrap Astral, we halved the measuring time in the mass spectrometer without impacting the depth of the redox proteome. We showed the applicability of SICyLIA-TMT to both cells in culture and full organs under stress condition. Hence, SICyLIA-cTMT is a versatile method that achieves unparalleled quantification accuracy and depth of the redox proteome while significantly reducing MS analysis time, making a significant step change in the study of oxidative signaling.

半胱氨酸氧化修饰（Cysteine oxidative modifications）是在生理与病理条件下调控细胞功能的核心信号事件。然而，由于其丰度较低，且难以全面、无偏倚地研究其动态变化，目前学界对这类修饰的认知仍十分有限。本研究开发了SICyLIA-TMT实验流程，该方法利用轻、重两种碘乙酰胺（IAA），对同一样品内的还原型与可逆氧化型半胱氨酸残基进行依次标记。通过将串联质量标签（TMTs）用于区分不同实验条件下的肽段标记，本策略仅需微克级起始样品，即可同时分析多组实验条件下的氧化修饰动态与蛋白质水平变化。此外，为提升对低丰度氧化型半胱氨酸残基的检测效能，我们设置了一条TMT通道作为重碘乙酰胺标记肽段的富集通道（SICyLIA-cTMT）。该策略显著提升了可逆氧化型半胱氨酸残基的定量效率，并可实现精准的化学计量比计算。最后，将SICyLIA-TMT与最新一代轨道阱Astral质谱仪（Orbitrap Astral）联用，我们将质谱仪的检测时长缩短了一半，且未对氧化蛋白质组（redox proteome）的覆盖深度造成负面影响。我们验证了SICyLIA-TMT在培养细胞与应激状态下完整器官中的适用性。综上，SICyLIA-cTMT是一种通用型方法，可实现前所未有的氧化蛋白质组定量精度与覆盖深度，同时大幅缩短质谱分析时长，为氧化信号通路研究带来了突破性进展。