Sulfo-NHS-SS-Biotin
(Synonyms: 1-[3-[[2-[[5-[(3AS,4S,6AR)-六氢-2-氧代-1H-噻吩并[3,4-D]咪唑-4-基]-1-氧代戊基]氨基]乙基]二硫基]-1-氧代丙氧基]-2,5-二氧代-3-吡咯烷磺酸钠,Biotin disulfide N-hydroxysulfosuccinimide ester) 目录号 : GC14824
Sulfo-NHS-SS-biotin 是一种胺反应性生物素化试剂。
Cas No.:325143-98-4
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
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- Purity: >98.00%
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- Datasheet
| MALDI-MS [1]: | |
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Principle |
Sulfo-NHS-SS-biotin as an alternative, versatile chemical derivatization agent for modification of primary amines prior to proteolysis, which allows for differential mass shifts of the same peptides, controlled by simple treatments of a single sample. |
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Applications |
Sulfo-NHS-SS-biotin crosslinker is an advantageous reagent for the analysis of lysine rich sequences by MALDI-MS because of its versatility. Prevention of trypsin cleavage after lysine residues leads to a higher sequence coverage and thus increases the chance to detect posttranslational modifications. |
| Cell experiment [2]: | |
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Cell lines |
MDA-MB-468 cell |
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Preparation Method |
Cells in monolayer culture were washed three times with ice-cold PBS and then treated with EZ-link sulfo-NHS-SS-biotin (1 mg/mL) for 15 minutes on ice. Biotinylation reactions were terminated with 100 mmol/L glycine in PBS. |
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Reaction Conditions |
1 mg/mL sulfo-NHS-SS-biotin for 15 minutes |
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Applications |
The cell surface abundance of ¦± integrin was examined by biotinylation( sulfo-NHS-SS-biotin ) and affinity precipitation. |
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References: [1]. Markoutsa S, Bahr U,et,al. Sulfo-NHS-SS-biotin derivatization: a versatile tool for MALDI mass analysis of PTMs in lysine-rich proteins. Proteomics. 2014 Mar;14(6):659-67. doi: 10.1002/pmic.201300309. PMID: 24449390. |
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Sulfo-NHS-SS-biotin is an amine-reactive biotinylating reagent.Sulfo-NHS-SS-biotin contains a negatively charged sulfonic acid group, giving it enough intramolecular polarity to be added directly to reactive aqueous solutions without the need for prior dissolution in organic solvents. Sulfo-ns-ss-biotin reacts with amine-containing proteins or other molecules to form a complex that further interacts with avidin or streptomycin probes to purify the target molecules by affinity chromatography[1].Sulfo-NHS-SS-biotin can be used for the biotinylation of plasma membrane proteins[5]. Amino groups on plasma membrane proteins are reversibly biotinylated by sulfo-NHS-SS-biotin. sulfo-NHS-SS-biotin contains a short spacer arm that includes a disulfide bond that can be cleaved by the addition of the membrane-impermeable reducing agent glutathione to cells[3,4].
Sulfo-NHS-SS-biotin crosslinker is an advantageous reagent for the analysis of lysine rich sequences by MALDI-MS because of its versatility. Prevention of trypsin cleavage after lysine residues leads to a higher sequence coverage and thus increases the chance to detect posttranslational modifications[2].
In MDA-MB-468 cell, The cell surface abundance of β1 integrin was examined by biotinylation(sulfo-NHS-SS-biotin ) and affinity precipitation[1]. To obtain a comprehensive view of the cell surface proteome of bone marrow-derived hMSCs,an analytical pipeline relying on cell surface biotinylation of intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin to enrich the plasma membrane proteins and mass spectrometry for identification with extremely high confidence. Among the 888 proteins identified, we found 200 bona fide plasma membrane proteins including 33 cell adhesion molecules and 26 signaling receptors. A significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells[6]. To localize the site(s) of action of DDM at the absorptive surface of Caco-2 cells, sulfo-NHS-SS-biotin, a membrane-impermeable compound, was applied apically. In the presence of 0.5 mM DDM, translocated biotin was found to be accumulated toward bicellular contacts, whereas no biotin permeation was observed in untreated control cells[7]. A dedicated protocol based on specific purification of surface membrane proteins labeled with sulfo-NHS-SS-biotin was developed. Appropriate gel electrophoresis separation and purification methods combined with standard proteomic methods were then used to identify and quantify surface membrane proteins from immature and mature spermatozoa. Membrane-associated proteins were discriminated from integral membrane proteins by differential solubilization. Protein regionalization on the spermatozoon surface was achieved by comparative analysis of the surface protein extracts from the entire spermatozoa and from periacrosomal sperm plasma membranes[8].
References:
[1]. Jo M, Eastman BM, et,al. Cell signaling by urokinase-type plasminogen activator receptor induces stem cell-like properties in breast cancer cells. Cancer Res. 2010 Nov 1;70(21):8948-58. doi: 10.1158/0008-5472.CAN-10-1936. Epub 2010 Oct 12. PMID: 20940399; PMCID: PMC2970644.
[2]. Markoutsa S, Bahr U, et,al. Sulfo-NHS-SS-biotin derivatization: a versatile tool for MALDI mass analysis of PTMs in lysine-rich proteins. Proteomics. 2014 Mar;14(6):659-67. doi: 10.1002/pmic.201300309. PMID: 24449390.
[3]. Walseng E, Furuta K, et,al. Ubiquitination regulates MHC class II-peptide complex retention and degradation in dendritic cells. Proceedings of the National Academy of Sciences of the United States of America. 2010 Nov;107(47):20465-20470. DOI: 10.1073/pnas.1010990107. PMID: 21059907; PMCID: PMC2996684.
[4]. Cho KJ, Walseng E, et,al. Ubiquitination by March-I prevents MHC class II recycling and promotes MHC class II turnover in antigen-presenting cells. Proc Natl Acad Sci U S A. 2015 Aug 18;112(33):10449-54. doi: 10.1073/pnas.1507981112. Epub 2015 Aug 3. PMID: 26240324; PMCID: PMC4547296.
[5]. Cho KJ, Roche PA. Monitoring MHC-II Endocytosis and Recycling Using Cell-Surface Protein Biotinylation-Based Assays. Methods Mol Biol. 2019;1988:271-277. doi: 10.1007/978-1-4939-9450-2_19. PMID: 31147946; PMCID: PMC8259318.
[6]. Niehage C, Steenblock C, et,al. The cell surface proteome of human mesenchymal stromal cells. PLoS One. 2011;6(5):e20399. doi: 10.1371/journal.pone.0020399. Epub 2011 May 26. PMID: 21637820; PMCID: PMC3102717.
[7]. Gradauer K, Iida M, et,al. Dodecylmaltoside Modulates Bicellular Tight Junction Contacts To Promote Enhanced Permeability. Mol Pharm. 2017 Dec 4;14(12):4734-4740. doi: 10.1021/acs.molpharmaceut.7b00297. Epub 2017 Oct 27. PMID: 28985076.
[8].Belleannee C, Belghazi M, et,al. Purification and identification of sperm surface proteins and changes during epididymal maturation. Proteomics. 2011 May;11(10):1952-64. doi: 10.1002/pmic.201000662. Epub 2011 Apr 7. PMID: 21472858.
Sulfo-NHS-SS-biotin 是一种胺反应性生物素化试剂。Sulfo-NHS-SS-biotin 含有带负电荷的磺酸基团，使其具有足够的分子内极性，可以直接添加到反应性水溶液中，而无需预先溶解在有机溶剂中。 Sulfo-ns-ss-biotin与含胺蛋白或其他分子反应形成复合物，进一步与亲和素或链霉素探针相互作用，通过亲和层析[1]纯化目标分子。Sulfo-NHS -SS-生物素可用于质膜蛋白的生物素化[5]。质膜蛋白上的氨基被磺基-NHS-SS-生物素可逆地生物素化。 sulfo-NHS-SS-biotin 包含一个短间隔臂，其中包含一个二硫键，该二硫键可以通过向细胞中添加不透膜的还原剂谷胱甘肽来裂解[3,4]。\n
Sulfo-NHS-SS-生物素交联剂因其多功能性而成为通过 MALDI-MS 分析富含赖氨酸序列的有利试剂。防止赖氨酸残基后的胰蛋白酶切割导致更高的序列覆盖度，从而增加检测翻译后修饰的机会[2]。
在MDA-MB-468细胞中，通过生物素化（sulfo-NHS-SS-biotin）和亲和沉淀法检测细胞表面β1整合素的丰度[1]。为了全面了解骨髓来源的 hMSCs 的细胞表面蛋白质组，分析管道依赖于完整细胞的细胞表面生物素化，使用细胞不可渗透、可裂解的磺基-NHS-SS-生物素来富集质膜蛋白和质谱以极高的信心识别。在鉴定的 888 种蛋白质中，我们发现了 200 种真正的质膜蛋白，包括 33 种细胞粘附分子和 26 种信号受体。 hMSCs细胞表面蛋白质组与人胚胎干细胞的显着差异[6]。为了在 Caco-2 细胞的吸收表面定位 DDM 的作用位点，在顶端应用了磺基-NHS-SS-生物素，一种膜不可渗透的化合物。在 0.5 mM DDM 的存在下，易位的生物素被发现向双细胞接触积累，而在未处理的对照细胞中未观察到生物素渗透[7]。开发了一种基于用磺基-NHS-SS-生物素标记的表面膜蛋白特异性纯化的专用方案。然后使用适当的凝胶电泳分离和纯化方法结合标准蛋白质组学方法来鉴定和量化来自未成熟和成熟精子的表面膜蛋白。通过差异增溶将膜相关蛋白与整合膜蛋白区分开来。通过比较分析整个精子和顶体周围精子质膜的表面蛋白提取物，实现精子表面蛋白区域化[8]。
| Cas No. | 325143-98-4 | SDF | |
| 别名 | 1-[3-[[2-[[5-[(3AS,4S,6AR)-六氢-2-氧代-1H-噻吩并[3,4-D]咪唑-4-基]-1-氧代戊基]氨基]乙基]二硫基]-1-氧代丙氧基]-2,5-二氧代-3-吡咯烷磺酸钠,Biotin disulfide N-hydroxysulfosuccinimide ester | ||
| 化学名 | sodium;1-[3-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyldisulfanyl]propanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate | ||
| Canonical SMILES | C1C(C(=O)N(C1=O)OC(=O)CCSSCCNC(=O)CCCCC2C3C(CS2)NC(=O)N3)S(=O)(=O)[O-].[Na+] | ||
| 分子式 | C19H27N4NaO9S4 | 分子量 | 606.7 |
| 溶解度 | ≥ 30.335mg/mL in DMSO ; <0.25mg/ml in H2O(Ultrasonic dissolution) | 储存条件 | Store at -20°C, unstable in solution, ready to use. |
| 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 | 1.6483 mL | 8.2413 mL | 16.4826 mL |
| 5 mM | 0.3297 mL | 1.6483 mL | 3.2965 mL |
| 10 mM | 0.1648 mL | 0.8241 mL | 1.6483 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。
Differential biotin labelling of the cell envelope proteins in lipopolysaccharidic diderm bacteria: Exploring the proteosurfaceome of Escherichia coli using Sulfo-NHS-SS-Biotin and sulfo-NHS-PEG4-bismannose-SS-biotin
J Proteomics 2018 Jun 15;181:16-23.29609094 10.1016/j.jprot.2018.03.026
Surface proteins are the major factor for the interaction between bacteria and its environment, playing an important role in infection, colonisation, virulence and adaptation. However, the study of surface proteins has proven difficult mainly due to their hydrophobicity and/or relatively low abundance compared with cytoplasmic proteins. To overcome these issues new proteomic strategies have been developed, such as cell-surface protein labelling using biotinylation reagents. Sulfo-NHS-SS-Biotin is the most commonly used reagent to investigate the proteins expressed at the cell surface of various organisms but its use in lipopolysaccharidic diderm bacteria (archetypical Gram-negative bacteria) remains limited to a handful of species. While generally pass over in silence, some periplasmic proteins, but also some inner membrane lipoproteins, integral membrane proteins and cytoplasmic proteins (cytoproteins) are systematically identified following this approach. To limit cell lysis and diffusion of the Sulfo-NHS-SS-Biotin through the outer membrane, biotin labelling was tested over short incubation times and proved to be as efficient for 1 min at room temperature. To further limit labelling of protein located below the outer membrane, the use of high-molecular weight sulfo-NHS-PEG4-bismannose-SS-biotin appeared to recover differentially cell-envelope proteins compared to low-molecular weight Sulfo-NHS-SS-Biotin. Actually, the Sulfo-NHS-SS-Biotin recovers at a higher extent the proteins completely or partly exposed in the periplasm than sulfo-NHS-PEG4-bismannose-SS-biotin, namely periplasmic and integral membrane proteins as well as inner membrane and outer membrane lipoproteins. These results highlight that protein labelling using biotinylation reagents of different sizes provides a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria. Significance: While generally pass over in silence, some periplasmic proteins, inner membrane lipoproteins (IMLs), integral membrane proteins (IMPs) and cytoplasmic proteins (cytoproteins) are systematically identified following cell-surface biotin labelling in lipopolysaccharidic diderm bacteria (archetypal Gram-negative bacteria). The use of biotinylation molecules of different sizes, namely Sulfo-NHS-SS-Biotin and sulfo-NHS-PEG4-bismannose-SS-biotin, was demonstrated to provide a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria.
Sulfo-NHS-SS-Biotin derivatization: a versatile tool for MALDI mass analysis of PTMs in lysine-rich proteins
Proteomics 2014 Mar;14(6):659-67.24449390 10.1002/pmic.201300309
The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of Sulfo-NHS-SS-Biotin derivatization of lysine side chain can help to detect PTMs in lysine-rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5-lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones.
Trafficking of immature DeltaF508-CFTR to the plasma membrane and its detection by biotinylation
Biochem J 2009 Apr 1;419(1):211-9, 2 p following 219.19053947 10.1042/BJ20081869
Recent studies suggest that immature, core-glycosylated DeltaF508-CFTR [the predominant mutant form of the CFTR (cystic fibrosis transmembrane conductance regulator)] can reach the plasma membrane under some conditions. In the present study we investigated this possibility since it has implications for understanding how therapeutics rescue the trafficking of mutant CFTR and perhaps other misfolded proteins. Core-glycosylated CFTR was labelled and pulled down on streptavidin beads after exposure to Sulfo-NHS-SS-Biotin [biotin attached to a reactive NHS (N-hydroxysuccinimide) ester with a disulfide spacer; molecular mass=606.7 Da]; however, intracellular proteins were also detected in the precipitates. When the R domain of CFTR was expressed in the cytosol of BHK (baby-hamster kidney) cells as a soluble polypeptide it was also labelled after surface biotinylation and pulled down on streptavidin beads. Intracellular biotinylation was reduced when cells were treated with sulfo-NHS-LC-biotin (biotin attached to a reactive NHS ester with an aminocaproic acid spacer) or sulfo-NHS-PEO(12)-biotin [biotin attached to a reactive NHS ester with a poly(ethylene glycol) spacer], but the reduction could be explained by the lower reactivity of these reagents. The R domain was detected on Western blots after loading <0.25% of the pulldown sample ( approximately 0.01% of total lysate protein), a fraction that could be ascribed to cells that were permeable to ethidium homodimer-1 (molecular mass=856.8 Da) and propidium iodide (molecular mass=668.6 Da). When BHK cells were incubated at 29 degrees C to rescue DeltaF508-CFTR trafficking, and then biotinylated and sorted to remove permeable cells, labelling of core-glycosylated DeltaF508-CFTR was no longer detected although a weak signal was still observed using CFBE (cystic fibrosis bronchial epithelial) cells. These results suggest that there is weak surface expression of immature DeltaF508-CFTR on airway epithelial cells and demonstrate the need to remove permeable cells when studying CFTR glycoforms by surface biotinylation.
Monitoring MHC-II Endocytosis and Recycling Using Cell-Surface Protein Biotinylation-Based Assays
Methods Mol Biol 2019;1988:271-277.31147946 PMC8259318
Most, if not all, plasma membrane proteins continuously undergo endocytosis and many rapidly recycle from endosomes back to the cell surface to maintain "stable" surface expression. We now describe a biochemical assay that is suited to follow the internalization and recycling kinetics of plasma membrane proteins. This assay involves biotinylation of plasma membrane proteins using Sulfo-NHS-SS-Biotin, a water-soluble, NHS-ester biotinylation reagent that contains a cleavable disulfide bond that allows for reversible labeling of proteins. Biotinylation is rapid and stable, and does not transfer from cell to cell, and the small size of the biotin probe does not affect cell function.
Surface-exposed proteins of Ehrlichia chaffeensis
Infect Immun 2007 Aug;75(8):3833-41.17517859 PMC1951975
The surface proteins of Ehrlichia chaffeensis provide an important interface for pathogen-host interactions. To investigate the surface proteins of E. chaffeensis, membrane-impermeable, cleavable Sulfo-NHS-SS-Biotin was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin-agarose affinity purification. The affinity-captured proteins were separated by electrophoresis, and five relatively abundant protein bands containing immunoreactive proteins were subjected to capillary-liquid chromatography-nanospray tandem mass spectrometry analysis. Nineteen out of 22 OMP-1/P28 family proteins, including P28 (which previously was shown to be surface exposed), were detected in E. chaffeensis cultured in human monocytic leukemia THP-1 cells. For the first time, with the exception of P28 and P28-1, 17 OMP-1/P28 family proteins were demonstrated to be expressed at the protein level. The surface exposure of OMP-1A and OMP-1N was verified by immunofluorescence microscopy. OMP-1B was undetectable either by surface biotinylation or by Western blotting of the whole bacterial lysate, suggesting that it is not expressed by E. chaffeensis cultured in THP-1 cells. Additional E. chaffeensis surface proteins detected were OMP85, hypothetical protein ECH_0525 (here named Esp73), immunodominant surface protein gp47, and 11 other proteins. The identification of E. chaffeensis surface-exposed proteins provides novel insights into the E. chaffeensis surface and lays the foundation for rational studies on pathogen-host interactions and vaccine development.