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]。