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Bioconjugation Technical Handbook

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Bioconjugation Technical Handbook Bioconjugation technical handbook Reagents for crosslinking, immobilization, modification, biotinylation, and fluorescent labeling of proteins and peptides Introduction Bioconjugation is the process of chemically joining two or more molecules or biomolecules by a covalent bond. This technique utilizes a variety of reagents for the crosslinking, immobilization, modification, and labeling of proteins and peptides. Bioconjugation reagents contain reactive ends to specific functional groups (e.g., primary amines, sulfhydryls) on proteins or other molecules. The availability of several chemical groups in proteins and peptides make them targets for a wide range of applications, including biotinylation, immobilization to solid supports, protein structural studies, and metabolic labeling. Chemical agents may be used to modify amino acid side chains on proteins and peptides in order to alter charges, block or expose reactive binding sites, inactivate functions, or change functional groups to create targets for crosslinking and labeling. Crosslinking, labeling, and modification reagents can be described by their chemical reactivity, molecular properties, or applications (Table 1). Table 1. Key considerations for selecting the right bioconjugation reagent. Chemical reactivity First, select a reagent with the functional group(s) to bind your biomolecules of interest. NHS ester reaction Maleimide reaction pH 6.5–7.5 Amine-containing NHS ester Amine bond NHS Sulfhydryl- Maleimide Thioether bond molecule compound containing compound molecule Molecular properties Second, choose which features or characteristics are important for your application. Functionality Cleavability Structural modifications OH O O O O O O N HO P N O O O O O DSS O OH NH2 HO O Disuccinimidyl suberate CA(PEG)TCEP n n MW 368.34 M.W. 250.15 DSS—same reactivity DSP with disulfide linker for cleavage TCEP—reducesCarboxy-PEG disulfiden-amine bonds Spacer arm 11.4 Å on both ends Carboxyl-(ethyleneglycol)n ethylamine n = 8 CA(PEG)8 Spacer arm composition Spacer arm length Spacer arm structure MW 441.51 18.1Å Spacer Arm 33.6Å O O O O O O n = 12 N O O CA(PEG)12 N N N HO O O NH 2 MW 617.72 O O O O Spacer Arm 46.8Å O CA(PEG)4 BMH AMAS MW 265.30 n = 24 M.W. 276.29 BMH with hydrocarbon spacer AMAS 4.4 Å—shortM.W. 252.18 spacer between CA(PEG)Spacern—adds Arm 18.1Å solubility in CA(PEG)24 Spacer Arm 16.1 Å reactiveSpacer Arm groups 4.4 Å aqueous solutions MW 1146.35 Applications Spacer Arm 89.8Å Select the specific reagent depending on the application (e.g., protein detection, immobilization, or interaction studies). Labeling Immobilization Protein interaction studies O O NH O O O HN O O O S O O N N N N O HO S O O O O O O O O DSS DSSO Disuccinimidyl sulfoxide Biotin—for labeling DSS—couplesDisuccinimidyl proteins sube ratote surfaces DSSO—MS-cleavable crosslinker MW 388.35 MW 368.34 and detection Spacer Arm 10.3 Å Spacer arm 11.4 Å Packaging options Select a package size or grade based on the scale of your reaction or your requirements. These reagents are available from milligram to kilogram quantities. Milligram Milligram to gram Milligram to gram Gram to kilogram Bulk-size packages available Single- tubes Catalog product Premium grade Large-volume or custom packages Contents Chemical reactivity of Hapten–carrier conjugation for bioconjugation reagents antibody production 27 Introduction 5 Protein–protein conjugation 28 Amine-reactive chemical groups 6 Creation of immunotoxins 29 Carboxylic acid–reactive Label transfer 29 chemical groups 7 Subunit crosslinking and protein Sulfhydryl-reactive chemical groups 8 structural studies 31 Carbonyl-reactive chemical groups 10 Protein interaction and crosslinking Nonspecificly-reactive chemical groups 12 using mass spectrometry 32 Chemoselective ligation 13 MS-cleavable crosslinkers 33 In vivo crosslinking 34 Molecular properties of Metabolic labeling 35 bioconjugation reagents Cell surface crosslinking 36 Introduction 14 Cell membrane structural studies 36 Homobifunctional and heterobifunctional crosslinkers 15 Special packaging to meet specific General reaction conditions 16 bioconjugation needs Modifications 16 Introduction 37 Spacer arm length 18 No-Weigh packaging format for Spacer arm composition 18 bioconjugation reagents 38 Spacer arm cleavability 18 Premium-grade bioconjugation reagents 39 Spacer arm structure and solubility 19 Bioconjugation resources 40 Applications using bioconjugation reagents Glossary 42 Introduction 20 Protein and peptide biotinylation 21 References 44 Fluorescent labeling of proteins and peptides 23 Ordering information 50 Protein immobilization Related handbooks and resources 71 onto solid supports 25 Surface modification using PEG-based reagents 26 Chemical reactivity of biconjugation reagents Introduction The most important property of a bioconjugation reagent is its reactive chemical group. The reactive group establishes the method and mechanism for chemical modification. Crosslinkers contain at least two reactive groups, which target common functional groups found in biomolecules such as proteins and nucleic acids. Protein modification reagents like PEGylation or biotinylation reagents have a reactive group at one terminus (PEG chain or biotin group, respectively) and a chemical moiety at the other end. The functional groups that are commonly targeted for bioconjugation include primary amines, sulfhydryls, carbonyls, carbohydrates, and carboxylic acids (Figure 1, Table 1). Coupling can also be nonselective using photoreactive groups. 5 O Amine-reactive chemical groups H2N O OH O H N H N 2 OH 2 OH Primary amines (–NH2) exist at the N-terminus of each R polypeptide chain (called the α-amine) and in the side chain HS of lysine (Lys, K) residues (called the ε-amine). Because of NH2 their positive charge at physiological conditions, primary Generic Lysine Cysteine amino acid amines are usually outward facing (i.e., on the outer surface of proteins), making them more accessible for conjugation without denaturing protein structure. A number of reactive O O O O O O O H H H H H H H3N N N N N N N chemical groups target primary amines (Figure 2), but the C C C C C C O R H H HO OH H most commonly used groups are N-hydroxysuccinimide SH esters (NHS esters) and imidoesters. O O HO O R N CS O O Isothiocyanate Cl R Gly Glu Gly Asp Gly Cys R S Cl R N NH C R N CO O H N Figure 1. Common amino acid functional groups targeted Isocyanate Sulfonyl chloride Aldehyde Carbodiimide O for bioconjugation. O O F O R Rʹ O O R N R N R O Carbonate Acyl azide Anhydride Fluorobenzene F O F F Table 2. Popular crosslinker reactive groups for protein conjugation. O Target N NH2 O HO R R O F R CH3 functional O O F Reactivity class group Reactive chemical group NHS ester Imidoester Epoxide Fluorophenyl ester Amine-reactive –NH2 NHS ester Imidoester Figure 2. Reactive chemical groups that target primary amines. Pentafluorophenyl ester Hydroxymethyl phosphine Carboxyl-to-amine reactive –COOH Carbodiimide (e.g., EDC) Sulfhydryl-reactive –SH Maleimide N-hydroxysuccinimide esters (NHS esters) Haloacetyl (bromo-, chloro-, or iodo-) Pyridyl disulfide O NHS esters are reactive groups formed by O Thiosulfonate N EDC activation of carboxylate molecules. O Vinyl sulfone R O NHS ester–activated crosslinkers and Aldehyde-reactive –CHO Hydrazide (e.g., oxidized Alkoxyamine NHS ester labeling compounds react with primary sugars, carbonyls) NHS ester amines in slightly alkaline conditions to Photoreactive Random Diazirine (i.e., nonselective, Aryl azide yield stable amide bonds (Figure 3). The reaction releases random insertion) N-hydroxysuccinimide, which can be removed easily by Hydroxyl (nonaqueous)- –OH Isocyanate dialysis or desalting. reactive Azide-reactive –N3 Alkyne Phosphine NHS-reactive chemistry NHS ester crosslinking reactions are most commonly performed in phosphate, carbonate–bicarbonate, HEPES, or borate buffers at pH 7.2–8.5 for 30 minutes to 4 hours at room temperature or 4°C. Primary amine buffers such as Tris (TBS) are not compatible, because they compete for the reaction. However, in some procedures it is useful to add Tris or glycine buffer at the end of a conjugation procedure to stop the reaction. Learn more at thermofisher.com/proteincrosslinking 6 O O O O Imidoester reaction chemistry P N NH N O 2 pH 7–9 N R + P R H + HO Imidoester crosslinkers react rapidly with amines at O O alkaline pH to form amidine bonds but have short half- NHS ester Primary amine Stable conjugate NHS reagent on protein (amide bond) lives. As the pH becomes more alkaline, the half-life and reactivity with amines increases, making crosslinking more Figure 3. NHS ester reaction scheme for chemical conjugation efficient when performed at pH 10 than at pH 8. Reaction to a primary amine. (R) represents a labeling reagent or one end of a crosslinker having the NHS ester reactive group; (P) represents a conditions below pH 10 may result in side reactions, protein or other molecule that contains the target functional group although amidine formation is favored between pH 8 and (i.e., primary amine). 10. Studies using monofunctional alkyl imidates reveal that at pH <10, conjugation can form with just one imidoester Hydrolysis of the NHS ester competes with the primary functional group. An intermediate N-alkyl imidate forms amine reaction. The rate of hydrolysis increases with buffer at the lower pH range and will either crosslink to another pH and contributes to less-efficient crosslinking in less- amine in the immediate vicinity, resulting in N,N´-amidine concentrated protein solutions. The half-life of hydrolysis derivatives, or it will convert to an amidine bond. At higher for NHS ester compounds is 4–5 hours at pH 7.0 and 0°C. pH, the amidine is formed directly without formation of This half-life decreases to 10 minutes at pH 8.6 and 4°C.
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