Disuccinimidyl Sulfoxide
(Synonyms: DSSO) 目录号 : GC43481Disuccinimidyl Sulfoxide（DSSO）是一种双功能、胺靶向、含亚砜的交联剂，用于通过交联质谱（XL-MS）分析蛋白质-蛋白质相互作用（PPI）。
Cas No.:1351828-03-9
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >95.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1. 溶液配制
（1）工作液：用DMF或DMO溶解Disuccinimidyl Sulfoxide，然后用实验缓冲液进行稀释，配制所需浓度的Disuccinimidyl Sulfoxide工作液。
注意：该产品的溶液形式不稳定，储存液和工作液建议现用现配，当天使用。最佳的工作浓度请根据实际情况调整。
2. 使用Disuccinimidyl Sulfoxide进行蛋白-蛋白交联[1](来源文献，仅供参考)
（1）Disuccinimidyl Sulfoxide与蛋白以60：1的摩尔过量进行交联反应，在室温下进行30min。
（2）用氯仿/甲醇沉淀蛋白质，并进行胰蛋白酶蛋白水解。
（3）通过StageTips净化胰蛋白酶肽溶液，并进行色谱分离，以富集交联肽。
（4）在质谱仪上分析。
注意：为了您的安全和健康，请穿实验服并戴一次性手套操作。
References:
[1]Singh R K, Soliman A, Guaitoli G, et al. Nanobodies as allosteric modulators of Parkinson’s disease–associated LRRK2[J]. Proceedings of the National Academy of Sciences, 2022, 119(9): e2112712119.
Disuccinimidyl Sulfoxide (DSSO) is a homobifunctional, amine-targeted, sulfoxide-containing cross-linker used for the analysis of protein-protein interactions (PPIs) by cross-linking mass spectrometry (XL-MS)[1, 2]. Disuccinimidyl Sulfoxide is suitable for model peptides and proteins as well as multi-subunit protein complexes[3]. Disuccinimidyl Sulfoxide contains two symmetrical collision-induced dissociation (CID) cleavable sites, which can effectively identify DSSO-cross-linked peptides based on different fragmentation patterns unique to the cross-link type[4]. The membrane-permeable Disuccinimidyl Sulfoxide spacer generates labeled peptides after cleavage, which can be unambiguously identified by collision-induced dissociation in XL-MS[5]. Disuccinimidyl Sulfoxide is a bifunctional N-hydroxysuccinimide-ester reagent with a spacer arm length of 10.1Å, which has the advantage of being able to fragment well in the gas phase[6]. Disuccinimidyl Sulfoxide reacts selectively with peptide amino groups in solution, thereby capturing adjacent lysine residues[6].
References:
[1] Kao A. Elucidating Elusive Structures: Determining 26S Proteasome Topology by Development of Novel High-throughput Cross-linking Mass Spectrometry Methodologies[M]. University of California, Irvine, 2013.
[2] Yu C, Novitsky E J, Cheng N W, et al. Exploring spacer arm structures for designs of asymmetric sulfoxide-containing MS-cleavable cross-linkers[J]. Analytical chemistry, 2020, 92(8): 6026-6033.
[3] Gutierrez C B. Expanding the Chemical Cross-linking Tool Kit for Cross-linking Mass Spectrometry[M]. University of California, Irvine, 2020.
[4] Kao A, Chiu C, Vellucci D, et al. Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes[J]. Molecular & Cellular Proteomics, 2011, 10(1).
[5] Chavez J D, Bruce J E. Chemical cross-linking with mass spectrometry: a tool for systems structural biology[J]. Current opinion in chemical biology, 2019, 48: 8-18.
[6] Fagerlund R D, Wilkinson M E, Klykov O, et al. Spacer capture and integration by a type IF Cas1–Cas2-3 CRISPR adaptation complex[J]. Proceedings of the National Academy of Sciences, 2017, 114(26): E5122-E5128.
Disuccinimidyl Sulfoxide（DSSO）是一种双功能、胺靶向、含亚砜的交联剂，用于通过交联质谱（XL-MS）分析蛋白质-蛋白质相互作用（PPI）[1, 2]。Disuccinimidyl Sulfoxide适用于模型肽和蛋白质以及多亚基蛋白质复合物[3]。Disuccinimidyl Sulfoxide含有两个对称的碰撞诱导解离（CID）可裂解位点，可根据交联类型独有的不同碎裂模式有效识别DSSO交联肽[4]。膜渗透性Disuccinimidyl Sulfoxide裂解后间隔区产生标记肽，能够通过XL-MS中的碰撞诱导解离进行明确识别[5]。Disuccinimidyl Sulfoxide是一种双官能N-羟基琥珀酰亚胺-酯试剂，具有10.1Å的间隔臂长度，优点是在气相中能够很好地片段化[6]。Disuccinimidyl Sulfoxide在溶液中选择性地与肽氨基反应，从而捕获紧邻的赖氨酸残基[6]。
| Cas No. | 1351828-03-9 | SDF | |
| 别名 | DSSO | ||
| 化学名 | 3,3'-sulfinylbis-propanoic acid, 1,1'-bis(2,5-dioxo-1-pyrrolidinyl) ester | ||
| Canonical SMILES | O=C(CCS(CCC(ON1C(CCC1=O)=O)=O)=O)ON2C(CCC2=O)=O | ||
| 分子式 | C14H16N2O9S | 分子量 | 388.3 |
| 溶解度 | DMF: 10 mg/ml DMSO: 20 mg/ml DMSO:PBS (pH 7.2)(1:5): 0.15 mg/ml | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.5753 mL | 12.8766 mL | 25.7533 mL |
| 5 mM | 0.5151 mL | 2.5753 mL | 5.1507 mL |
| 10 mM | 0.2575 mL | 1.2877 mL | 2.5753 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Generation of Antibodies Targeting Cleavable Cross-Linkers
Anal Chem 2021 Mar 2;93(8):3762-3769.PMID:33591729DOI:10.1021/acs.analchem.0c04043.
Chemical cross-linking has become a powerful tool for the analysis of protein structures and interactions by mass spectrometry. A particular strength of this approach is the ability to investigate native states in vivo, investigating intact organelles, cells, or tissues. For such applications, the cleavable cross-linkers Disuccinimidyl Sulfoxide (DSSO) and disuccinimidyl dibutyric urea (DSBU) are gaining increasing popularity, as they allow for the analysis of complex mixtures. It is inherently difficult to follow the reaction of cross-linkers with proteins in intact biological structures, stalling the optimization of in vivo cross-linking experiments. We generated polyclonal antibodies targeting DSSO- and DSBU-modified proteins, by injection of cross-linked bovine serum albumin (BSA) in rabbits. We show that the cross-linker-modified BSA successfully triggered an immune response, and that DSSO- and DSBU-specific antibodies were generated by the animals. Using affinity-purified antibodies specific for the individual cross-linkers, we demonstrate their application to the detection of cross-linker-modified proteins in Western blot and immunocytochemistry experiments of intact and permeabilized cells. Furthermore, we show their ability to immunoprecipitate DSSO/DSBU-modified proteins and provide evidence for their affinity toward water-quenched dead-links. These antibodies provide a valuable tool for the investigation of proteins modified with the cross-linkers DSSO and DSBU.
Exploring Spacer Arm Structures for Designs of Asymmetric Sulfoxide-Containing MS-Cleavable Cross-Linkers
Anal Chem 2020 Apr 21;92(8):6026-6033.PMID:32202417DOI:10.1021/acs.analchem.0c00298.
Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for defining protein-protein interactions (PPIs) and elucidating architectures of large protein assemblies. To advance XL-MS studies, we have previously developed a series of sulfoxide-containing MS-cleavable cross-linkers to facilitate the detection and identification of cross-linked peptides using multistage mass spectrometry (MSn). While current sulfoxide-based cross-linkers are effective for in vivo and in vitro XL-MS studies at the systems-level, new reagents are still needed to help expand PPI coverage. To this end, we have designed and synthesized six variable-length derivatives of Disuccinimidyl Sulfoxide (DSSO) to better understand the effects of spacer arm modulation on MS-cleavability, fragmentation characteristics, and MS identification of cross-linked peptides. In addition, the impact on cross-linking reactivity was evaluated. Moreover, alternative MS2-based workflows were explored to determine their feasibility for analyzing new sulfoxide-containing cross-linked products. Based on the results of synthetic peptides and a model protein, we have further demonstrated the robustness and predictability of sulfoxide chemistry in designing MS-cleavable cross-linkers. Importantly, we have identified a unique asymmetric design that exhibits preferential fragmentation of cross-links over peptide backbones, a desired feature for MSn analysis. This work has established a solid foundation for further development of sulfoxide-containing MS-cleavable cross-linkers with new functionalities.
Developing a Targeted Quantitative Strategy for Sulfoxide-Containing MS-Cleavable Cross-Linked Peptides to Probe Conformational Dynamics of Protein Complexes
Anal Chem 2022 Mar 15;94(10):4390-4398.PMID:35193351DOI:10.1021/acs.analchem.1c05298.
In recent years, cross-linking mass spectrometry (XL-MS) has made enormous strides as a technology for probing protein-protein interactions (PPIs) and elucidating architectures of multisubunit assemblies. To define conformational and interaction dynamics of protein complexes under different physiological conditions, various quantitative cross-linking mass spectrometry (QXL-MS) strategies based on stable isotope labeling have been developed. These QXL-MS approaches have effectively allowed comparative analysis of cross-links to determine their relative abundance changes at global scales. Although successful, it remains challenging to consistently obtain quantitative measurements on low-abundant cross-links. Therefore, targeted QXL-MS is needed to enable MS "Western" analysis of cross-links to enhance sensitivity and reliability in quantitation. To this end, we have established a robust parallel reaction monitoring (PRM)-based targeted QXL-MS platform using sulfoxide-containing MS-cleavable cross-linker Disuccinimidyl Sulfoxide (DSSO), permitting label-free comparative analysis of selected cross-links across multiple samples. In addition, we have applied this methodology to study phosphorylation-dependent conformational dynamics of the human 26S proteasome. The PRM-based targeted QXL-MS analytical platform described here is applicable for all sulfoxide-containing MS-cleavable cross-linkers and can be directly adopted for comparative studies of protein-protein interactions in various cellular contexts.
Improved Peptide Backbone Fragmentation Is the Primary Advantage of MS-Cleavable Crosslinkers
Anal Chem 2022 Jun 7;94(22):7779-7786.PMID:35613060DOI:10.1021/acs.analchem.1c05266.
Proteome-wide crosslinking mass spectrometry studies have coincided with the advent of mass spectrometry (MS)-cleavable crosslinkers that can reveal the individual masses of the two crosslinked peptides. However, recently, such studies have also been published with noncleavable crosslinkers, suggesting that MS-cleavability is not essential. We therefore examined in detail the advantages and disadvantages of using the commonly used MS-cleavable crosslinker, Disuccinimidyl Sulfoxide (DSSO). Indeed, DSSO gave rise to signature peptide fragments with a distinct mass difference (doublet) for nearly all identified crosslinked peptides. Surprisingly, we could show that it was not these peptide masses that proved the main advantage of MS cleavability of the crosslinker, but improved peptide backbone fragmentation which reduces the ambiguity of peptide identifications. This also holds true for another commonly used MS-cleavable crosslinker, DSBU. We show furthermore that the more intricate MS3-based data acquisition approaches lack sensitivity and specificity, causing them to be outperformed by the simpler and faster stepped higher-energy collisional dissociation (HCD) method. This understanding will guide future developments and applications of proteome-wide crosslinking mass spectrometry.
3-Dimensional architecture of the human multi-tRNA synthetase complex
Nucleic Acids Res 2020 Sep 4;48(15):8740-8754.PMID:32644155DOI:10.1093/nar/gkaa569.
In mammalian cells, eight cytoplasmic aminoacyl-tRNA synthetases (AARS), and three non-synthetase proteins, reside in a large multi-tRNA synthetase complex (MSC). AARSs have critical roles in interpretation of the genetic code during protein synthesis, and in non-canonical functions unrelated to translation. Nonetheless, the structure and function of the MSC remain unclear. Partial or complete crystal structures of all MSC constituents have been reported; however, the structure of the holo-MSC has not been resolved. We have taken advantage of cross-linking mass spectrometry (XL-MS) and molecular docking to interrogate the three-dimensional architecture of the MSC in human HEK293T cells. The XL-MS approach uniquely provides structural information on flexibly appended domains, characteristic of nearly all MSC constituents. Using the MS-cleavable cross-linker, Disuccinimidyl Sulfoxide, inter-protein cross-links spanning all MSC constituents were observed, including cross-links between eight protein pairs not previously known to interact. Intra-protein cross-links defined new structural relationships between domains in several constituents. Unexpectedly, an asymmetric AARS distribution was observed featuring a clustering of tRNA anti-codon binding domains on one MSC face. Possibly, the non-uniform localization improves efficiency of delivery of charged tRNA's to an interacting ribosome during translation. In summary, we show a highly compact, 3D structural model of the human holo-MSC.