Department of Physiology and Biophysics, University of California, Irvine, Irvine, California 92697, United States.
Department of Chemistry, University of California, Irvine, Irvine, California 92697, United States.
Anal Chem. 2023 Jan 31;95(4):2532-2539. doi: 10.1021/acs.analchem.2c04986. Epub 2023 Jan 18.
The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein-protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide (BMSO)) that is effective for mapping PPIs of protein complexes to yield interaction contacts complementary to lysine-reactive reagents. While successful, the hydrolysis and limited selectivity of maleimides at physiological pH make their applications in proteome-wide XL-MS challenging. To enable global PPI mapping, we have explored an alternative cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide (DBrASO). Our results have demonstrated that DBrASO cross-linked peptides display the same fragmentation characteristics as other sulfoxide-containing MS-cleavable cross-linkers, permitting their unambiguous identification by MS. In combination with a newly developed two-dimensional peptide fractionation method, we have successfully performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents and expand PPI coverage at the systems-level.
MS 可切割交联质谱 (XL-MS) 的发展使人们能够在无需细胞工程的情况下，有效地捕获和鉴定内源性蛋白质-蛋白质相互作用 (PPIs) 及其残基接触，达到全局规模。到目前为止，只有赖氨酸反应性交联剂已成功应用于全蛋白质组 PPI 分析。然而，单独的赖氨酸交联剂无法揭示细胞中完整的 PPI 图谱。此前，我们开发了一种基于马来酰亚胺的半胱氨酸反应性 MS 可切割交联剂（双马来酰亚胺亚砜 (BMSO)），该交联剂可有效绘制蛋白质复合物的 PPIs，产生与赖氨酸反应性试剂互补的相互作用接触。虽然成功了，但马来酰亚胺在生理 pH 下的水解和有限的选择性使其在全蛋白质组 XL-MS 中的应用具有挑战性。为了实现全局 PPI 作图，我们探索了另一种半胱氨酸标记化学方法，因此设计并合成了一种含亚砜的 MS 可切割卤代乙酰胺基交联剂，二溴乙酰胺亚砜 (DBrASO)。我们的结果表明，DBrASO 交联肽显示出与其他含亚砜的 MS 可切割交联剂相同的片段特征，允许通过 MS 对其进行明确鉴定。结合新开发的二维肽分级方法，我们成功地对 HEK293 细胞裂解物进行了基于 DBrASO 的 XL-MS 分析，并证明了其补充赖氨酸反应性试剂和扩大系统水平 PPI 覆盖范围的能力。
