DTSSP Crosslinker - CAS 81069-02-5

DTSSP Crosslinker - CAS 81069-02-5 Catalog number: BADC-01163

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Category
ADCs Linker
Product Name
DTSSP Crosslinker
CAS
81069-02-5
Catalog Number
BADC-01163
Molecular Formula
C14H16N2O14S4
Molecular Weight
564.54
DTSSP Crosslinker

Ordering Information

Catalog Number Size Price Quantity
BADC-01163 -- $--
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Synonyms
3,3'-Dithiobis(sulphosuccinimidyl propionate); 3-pyrrolidinesulfonic acid, 1,1'-[dithiobis[(1-oxo-3,1-propanediyl)oxy]]bis[2,5-dioxo-
Canonical SMILES
C1C(C(=O)N(C1=O)OC(=O)CCSSCCC(=O)ON2C(=O)CC(C2=O)S(=O)(=O)O)S(=O)(=O)O
InChI
InChI=1S/C14H16N2O14S4/c17-9-5-7(33(23,24)25)13(21)15(9)29-11(19)1-3-31-32-4-2-12(20)30-16-10(18)6-8(14(16)22)34(26,27)28/h7-8H,1-6H2,(H,23,24,25)(H,26,27,28)
InChIKey
VOTJUWBJENROFB-UHFFFAOYSA-N
Density
1.940 g/cm3 (Predicted)
Solubility
10 mm in DMSO
Melting Point
> 222 °C (dec.)
Index Of Refraction
1.693
LogP
0.13240
PSA
303.46000
Appearance
Off-white to light tan solid
Purity
≥97.0%
Quantity
Data not available, please inquire.
Shelf Life
-20°C 3 years powder; -80°C 2 years in solvent
Shipping
Room temperature
Storage
Store at -20 °C, keep in dry and avoid sunlight.
Pictograms
Irritant
Signal Word
Warning
Form
Solid

DTSSP, or 3,3’-dithiobis(sulfosuccinimidyl propionate), is a widely used water-soluble crosslinker that features amine-reactive NHS-ester groups on both ends of an 8-atom spacer arm. The central disulfide bond in this spacer can be cleaved using reducing agents, making DTSSP particularly versatile in biochemical applications. The sulfo-NHS esters of DTSSP react specifically with primary amines, found commonly in the side chains of lysine residues or the N-terminus of protein polypeptides. This reactivity allows DTSSP to form stable amide bonds, providing a reliable means to link proteins, including antibodies. The cleavable nature of DTSSP’s spacer arm offers a key advantage for applications that require the eventual separation of crosslinked products, setting it apart from non-cleavable crosslinkers like its non-sulfonated analog, DSS.

In drug discovery, DTSSP’s capabilities extend to the study of protein interactions and structural biology. By crosslinking primary amines on protein surfaces, researchers can stabilize weak and transient interactions that might otherwise escape detection. This stabilization allows for the identification and characterization of such interactions, which are crucial for understanding biological pathways and mechanisms of action for potential therapeutics. Additionally, DTSSP is instrumental in immobilizing proteins onto amine-coated surfaces, thus facilitating the development of assays that can screen for drug activity or detect biomarkers. Its success in forming bioconjugates is due largely to its efficiency in creating single-step chemical crosslinks, an important factor in high-throughput screening environments.

The water solubility of DTSSP ensures that it is particularly useful for applications involving cell surface labeling. Its membrane-impermeable nature means it primarily targets extracellular proteins, thus serving as a valuable tool for capturing cell surface interactions prior to cell lysis and further analysis such as immunoprecipitation. This feature is crucial in the study of membrane proteins, which are often integral to cell signaling and are popular targets for drug development. Given that membrane proteins frequently have roles in disease pathways, the ability to study them effectively using a crosslinker like DTSSP reinforces its importance in pharmacological research.

DTSSP also finds application in improving the specificity and reliability of bioconjugates in proteomics. Its specifications, including a purity standard of over 80% as confirmed by quantitative NMR, ensure consistent performance in complex biological systems. Each lot of DTSSP is manufactured to meet stringent quality controls, providing researchers with confidence in their experimental outputs. The ability to cleave the crosslinker post-reaction simplifies the analysis process, allowing for clearer interpretation of mass spectrometry data and enhancing the identification of novel protein interactions or modifications. Such precision is indispensable in identifying drug targets and understanding drug effects at the molecular level.

1.Understanding chemical reactivity for homo- and heterobifunctional protein cross-linking agents.
Chen F1, Nielsen S, Zenobi R. J Mass Spectrom. 2013 Jul;48(7):807-12. doi: 10.1002/jms.3224.
Chemical cross-linking, combined with mass spectrometry, has been applied to map three-dimensional protein structures and protein-protein interactions. Proper choice of the cross-linking agent, including its reactive groups and spacer arm length, is of great importance. However, studies to understand the details of reactivity of the chemical cross-linkers with proteins are quite sparse. In this study, we investigated chemical cross-linking from the aspects of the protein structures and the cross-linking reagents involved, by using two structurally well-known proteins, glyceraldehyde 3-phosohate dehydrogenase and ribonuclease S. Chemical cross-linking reactivity was compared using a series of homo- and hetero-bifunctional cross-linkers, including bis(sulfosuccinimidyl) suberate, dissuccinimidyl suberate, bis(succinimidyl) penta (ethylene glycol), bis(succinimidyl) nona (ethylene glycol), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, 2-pyridyldithiol-tetraoxaoctatriacontane-N-hydrosuccinimide and succinimidyl-[(N-maleimidopropionamido)-tetracosaethyleneglycol]ester.
2.Localization of a site of intermolecular cross-linking in human red blood cell band 3 protein.
Jennings ML, Nicknish JS. J Biol Chem. 1985 May 10;260(9):5472-9.
Subunit interactions in the band 3 protein of the human red blood cell membrane have been examined by a combination of cross-linking, chemical labeling, and in situ proteolysis. In agreement with Staros (Staros, J. V. (1982) Biochemistry 21, 3950-3955), we find that the membrane-impermeant active ester bis(sulfosuccinimidyl) suberate (BSSS) cross-links band 3 in intact cells to a dimer, with no formation of higher oligomer. Combined cross-linking of the outer surface with BSSS and the cytoplasmic domain with Cu2+/o-phenanthroline does not produce significant covalent tetramer of band 3 (beyond that produced by Cu2+/o-phenanthroline alone). Therefore, the membrane domains and cytoplasmic domains of the same pair of subunits are cross-linked to each other. 4,4'-Diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) is known to form a covalent cross-link between complementary chymotryptic fragments (Mr 60,000 and 35,000). Edman degradation of band 3 from H2DIDS/chymotrypsin-treated cells shows that the H2DIDS cross-link is between fragments of the same subunit.
3.Intramolecular cross-linking of oxy hemoglobin by bis sulfosuccinimidyl suberate and sebacate: generation of cross-linked hemoglobin with reduced oxygen affinity.
Manjula BN1, Smith PK, Malavalli A, Acharya AS. Artif Cells Blood Substit Immobil Biotechnol. 1995;23(3):311-8.
The sulfosuccinimidyl esters of suberic and sebacic acids readily introduce intramolecular crosslinks into oxy HbA at pH 7.4, the relative efficiency of crosslinking by the suberate ester being slightly higher than that of sebacate. Nearly quantitative intramolecular crosslinking of HbA (0.5 mM) is achieved at pH 7.4 and 4 degrees C by using 5 and 10 fold molar excess of the suberic and sebacic acid, respectively. In contrast to the facile crosslinking reaction seen with the bis sulfosuccinimidyl sebacate, bis sulfosuccinimidyl sebacate and bis (3:5 dibromo salicyl) sebacate did not introduce any crosslinking into HbA despite the fact that the 'crosslinking arm' of the two bifunctional reagents is the same. The discrepant reactivity of the two reagents demonstrates the 'steering' influence of the negative charge of the leaving group of the reagent, namely sulfo succinimidyl moiety to specific domains of HbA rich in positively charged groups.
4.Distance restraints from crosslinking mass spectrometry: mining a molecular dynamics simulation database to evaluate lysine-lysine distances.
Merkley ED1, Rysavy S, Kahraman A, Hafen RP, Daggett V, Adkins JN. Protein Sci. 2014 Jun;23(6):747-59. doi: 10.1002/pro.2458. Epub 2014 Apr 3.
Integrative structural biology attempts to model the structures of protein complexes that are challenging or intractable by classical structural methods (due to size, dynamics, or heterogeneity) by combining computational structural modeling with data from experimental methods. One such experimental method is chemical crosslinking mass spectrometry (XL-MS), in which protein complexes are crosslinked and characterized using liquid chromatography-mass spectrometry to pinpoint specific amino acid residues in close structural proximity. The commonly used lysine-reactive N-hydroxysuccinimide ester reagents disuccinimidylsuberate (DSS) and bis(sulfosuccinimidyl)suberate (BS(3) ) have a linker arm that is 11.4 Å long when fully extended, allowing Cα (alpha carbon of protein backbone) atoms of crosslinked lysine residues to be up to ∼24 Å apart. However, XL-MS studies on proteins of known structure frequently report crosslinks that exceed this distance.
The molarity calculator equation

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

The dilution calculator equation

Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2

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