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Deuterated Crosslinking Reagents for Mass Spec Analysis |
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Deuterated Crosslinking Agents and Mass Spectrometry Prove to be a Powerful New Combination in Protein Structure-Function Analysis.
Use of new crosslinker pairs can deliver protein folding information or protein:protein interaction mapping data
Chemical crosslinking reagents have long been used as tools to probe protein structure and gain insight into protein function. Pierce Double-Agents crosslinking reagents have played an important role in the studies published to date. A new strategy to study protein structure and function is rapidly emerging that integrates the proven utility of crosslinking with the power of mass spectrometry (MS) to yield insights into protein tertiary structure and protein complex formation.1 High quality "heavy" and "light" crosslinkers analogs are now available to catalyze the potential of this method. Pierce continues its leadership in protein crosslinking by synthesizing and characterizing the deuterated analogs of BS2G and BS3 for the mass spectral study of intramolecular and intermolecular interactions.
| What is a heavy crosslinker? |
Why are heavy crosslinkers key to structure-function analysis by MS? |
- A heavy crosslinker is the deuterated analog of a specific crosslinking agent. In a heavy crosslinker, D (deuterium) is substituted for H (hydrogen) on specific methylene carbons located within the crosslinker hydrocarbon spacer arm.
- For reagents introduced here, -CH2- is converted to a -CD2- on 2 methylene carbons in their respective hydrocarbon spacer arms.
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- Peptide masses differing by 4 units unambiguously identify a crosslink at a specific locus within a protein structure or between protein binding partners.
- Crosslinker spacer arm length can be used to assign distance constraints between crosslinked peptides.
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More about heavy and light crosslinking reagent pairs and mass spectrometric analysis
Heavy (-dn) and light (-d0) crosslinking reagent analogs are reacted simultaneously with the target protein or protein complex. Incorporating deuterium at discrete positions in the crosslinking reagent allows both protein crosslinking and protein labeling to occur in a single step. Use of heavy/light crosslinker pairs in this application simplifies the MS identification of the peptides resulting from the coupling reactions. Application of a 1:1 ratio of two identical crosslinking agents differing only in the number of deuterium atoms in their chemical composition (e.g. d4 vs d0) is a powerful identifier of low abundant singly crosslinked peptides in the mass spectrometer. The reagents introduced here will generate MS patterns in which the resultant crosslinked peptides will differ by 4 mass units after enzymatic digestion of the crosslinked protein or protein complex. Further analysis of the reaction products can yield low resolution three dimensional structure information. Intermolecular crosslinking of an interacting protein complex and MS analysis have been successfully applied to determine the molecular contact surfaces of binding partners in a protein complex.2-6
Heavy and light crosslinker pairs developed for MS applications.
The reagents described here, prepared specifically for these MS applications, are the light and heavy analogs of BS2G and BS3. These are homobifunctional, amine-reactive, non-cleavable, water soluble crosslinking agents with defined spacer arm lengths. These reagents can act as molecular rulers for estimation of spatial relationships in protein structure-function studies. Both the light analogs (BS2G-d0 and BS3-d0) and the deuterated analogs (BS2G-d4 and BS3-d4) react efficiently with primary amino groups (-NH2) at pH 7-9 to form stable amide bonds. Proteins generally contain several primary amines in the side chain of Lysine (K) residues and the N-terminus of each polypeptide that are available as targets for the two sulfo-NHS ester reactive groups found in each of these reagents. These reagents are supplied as a sodium salt and are soluble in water at a concentration up to 10 mM.
| FEATURES |
BENEFITS |
- Well characterized, high purity, deuterium-labeled crosslinkers and their hydrogen-containing analogs.
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- Reagents specially prepared and analyzed for use with this crosslinking - mass spectrometry strategy for protein structure:function analysis
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- Instructions targeted to the application of structure and interaction analysis
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- Instructions, edited by an expert in this MS-based method, help guide the first time user through the application.
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- Suberate and glutarate-based reagent pairs offer a "molecular ruler" option to study inter- and intra-molecular distances.
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- A single protein can be "measured" with two distinct reagents to gain additional structural information not possible with just one reagent.
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- Only microgram amounts of protein are required to perform the analysis.
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- Excellent alternative to NMR or X-ray crystallography-based methods that require large amounts of protein, special solvents or crystal formation.
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General Protocol for Tertiary Structure and Protein Interaction Applications
MS-based applications using deuterated BS3 and BS2G
Application #1
Application #2
References Illustrating Applications of Heavy/Light Crosslinking Reagents and Mass Spectrometry
- Sinz, A. (2003). Chemical crosslinking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes. J. Mass Spectrom. 38, 1225-1237.
- Dihazi, G.H. and Sinz, A. (2003). Mapping low-resolution three-dimensional protein structure using chemical crosslinking and Fourier transform ion-cyclotron resonance mass spectrometry. Rapid Commun. Mass Spectrom. 17, 2005-2014.
- Kalkhof, S. et al. (2005). Chemical Crosslinking and High-Performance Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Protein Interaction Analysis: Application to a Calmodulin/Target Peptide Complex. Anal. Chem. 77, 495-503.
- Back, J.W. et al. (2003). Chemical Crosslinking and mass spectrometry for protein structural modeling. J. Mol. Biol. 331, 303-313.
- Pearson, K. M., et al. (2002). Intramolecular crosslinking experiments on cytochrome c and ribonuclease A using an isotope multiplet method. Rapid Commun. Mass Spectrom. 16, 149-159.
- Muller, D.R. et al. (2001). Isotope-tagged crosslinking reagents. A new tool in mass spectrometric protein interaction analysis. Anal. Chem. 73, 1927-1934
- Schilling, B. et al. (2003). MS2 assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides. J. Am. Soc. Mass Spectrom. 14, 834-850.
- Peri, S. et al. (2001). GPMAW- a software tool for analyzing proteins and peptides. Trends Biochem. Sci. 26, 687-689.
See also the 2005/'06 Pierce Applications Handbook and Catalog, pp. 412, 434-435.
Related Products
AQUA and Heavy Peptides
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Ordering Information
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 Bulk quantities: Inquire  Certificate
of Analysis  Product
Instructions  MSDS |
| Product # |
Description |
Pkg. Size |
Files |
Price |
| 21590 |
BS3-d0
(Bis[Sulfosuccinimidyl] suberate-d0) |
10 mg |
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$58.00
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| 21595 |
BS3-d4
(Bis[Sulfosuccinimidyl] 2,2,7,7-suberate-d4) |
10 mg |
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$202.00
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| 21610 |
BS2G-d0
(Bis[Sulfosuccinimidyl] glutarate-d0) |
10 mg |
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$57.00
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| 21615 |
BS2G-d4
(Bis[Sulfosuccinimidyl] 2,2,4,4-glutarate-d4) |
10 mg |
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$205.00
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