Biotinylation of Peptides/Proteins using Biocytin Hydrazide

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544 J. Chin. Chem. Soc., Vol. 54, No. 2, 2007 Lee et al. Table 1. Observed molecular masses of peptides and proteins as well as their biotinylated ions produced by reacting peptides/proteins with biocytin hydrazide (BH) and other hydrazide derivatives Proteins/Peptides Incubation conditions Mass (m/z) 0.1% No. of Biotin or Hydrazide derivative attached Angiotensin I 65 C, 24 h, 10% TFA 1297 0 Angiotensin I 65 C, 24 h, 10% TFA, 1 M BH 1667 1 P14R 65 C, 24 h, 10% TFA 1534 0 P14R 65 C, 24 h, 10% TFA, 1 M BH 1904 1 ACTH Fragment 1-13 65 C, 24 h, 10% TFA 1624 0 ACTH Fragment 1-13 65 C, 24 h, 10% TFA, 1 M BH 1994 1 Insulin 65 C, 24 h, PBS 5735 0 Insulin 0.3 M Biocytin HCl a 5735 0 Insulin 0.3 M Phenyl hydrazine a 5826, 5917 1, 2 Insulin 0.3 M Adipic Dihydrazide a 5892, 6049 1, 2 Insulin 0.3 M 2-Iminobiotin a 5735 0 Insulin 0.3 M 2-Iminobiotin Hydrazide a and 91 Da upon condensation with biocytin HCl, 2-iminobiotin hydrazide, 2-iminobiotin, adipic dihydrazide, and phenyl hydrazine, respectively. Data shows that biotinylated insulins are detected at m/z 5976 (singly biotinylated) and m/z 6217 (doubly biotinylated) using 2-iminobiotin hydrazide as the biotinylation reagent, at m/z 5892 (singly 5976, 6217 1, 2 Insulin 65 C, 4 h, 10% TFA, 1 M BH 6105, 6475 1, 2 Insulin 65 C, 5 h, 10% TFA, 0.01 M BH 5735 0 Insulin 65 C, 5 h, 10% TFA, 0.1 M BH 6105 1 Insulin 65 C, 5 h, 10% TFA, 1 M BH 6105, 6475 1, 2 Insulin 4 C, 5 h, 10% TFA, 1 M BH 5735 0 Insulin 37 C, 5 h, 10% TFA, 1 M BH 6105 1 Insulin 65 C, 24 h, 10% TFA, 1 M BH 6105, 6475, 6845, 7215, 7585 1, 2, 3, 4, 5 Insulin 65 C, 24 h, pH 10 b ,1M BH 6105, 6475, 6845, 7215, 1, 2, 3, 4 Insulin 65 C, 24 h, PBS, 1 M BH 6105, 6475, 6845, 7215, 7585, 7955, 8325 1, 2, 3, 4, 5, 6, 7 Cytochrome c 37 C, 96 h, 10% TFA 12362 0 Cytochrome c 37 C, 96 h, 10% TFA, 0.3 M BH 12732, 13102, 13472, 13842, 14212, 14582 1, 2, 3, 4, 5, 6 Lysozyme 65 C, 19 h, 10% TFA 14306 0 Lysozyme 65 C, 4 h, 10% TFA, 1 M BH 14676, 15046 1, 2 Lysozyme 65 C, 19 h, 10% TFA, 1 M BH 14676, 15046, 15416, 15786, 16156, 16526 1, 2, 3, 4, 5, 6 SBTI 65 C, 19 h, PBS 19995 0 SBTI 65 C, 19 h, PBS, 1 M BH 20365, 20735, 21105, 21475 1, 2, 3, 4 BSA 65 C, 19 h, PBS 66430 0 BSA 65 C, 5 h, PBS, 0.01 M BH 66939 1.4 BSA 65 C, 5 h, PBS, 0.1 M BH 67042 1.7 BSA 65 C, 5 h, PBS, 1 M BH 72600 17 BSA 65 C, 5 h, 10% TFA, 1 M BH 67712, 70793 3.5, 12 BSA 65 C, 5 h, pH 10 b ,1M BH 68874 6.6 BSA 4 C, 5 h, PBS, 1 M BH 68813 6.4 BSA 37 C, 5 h, PBS, 1 M BH 69600 8.6 BSA 65 C, 19 h, PBS, 1 M BH 72720, 83450 17, 46 a Reaction was done at 65 C for 5.5 h. in aqueous solution of 50% acetonitrile (v/v) containing 1% TFA (v/v). b 50 mM Na2CO3 aqueous solution at pH 10. Zip-Tips (Millipore, Billerica, MA) packed with C18 and C4 resin were used to prepare the solution for MS analysis of peptides (1 g/l) and proteins (1 g/l), respectively. Cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid (SA) were used as the matrix for peptides and proteins, respectively. biotinylated) and m/z 6049 (doubly biotinylated) using adipic dihydrazide as the biotinylation reagent, and at m/z 5826 (singly biotinylated) and m/z 5917 (doubly biotinylated) using phenyl hydrazine as the biotinylation reagent. However, no biotinylated insulin is detected using either biocytin HCl or 2-iminobiotin as the biotinylation reagent.
Biotinylation of Peptides/Proteins using Biocytin Hydrazide J. Chin. Chem. Soc., Vol. 54, No. 2, 2007 545 It is noted that the cross-linking ion between two insulins and one adipic dihydrazide was detected at m/z 11609 (data not shown). Results indicate that it is the hydrazide functional group of the biotinylated reagents that is responsible for the biotinylation reaction. Compared to the traditional biotinylation reagents such as N-hydroxysuccinimide (NHS)biotin (NH2 reactive), maleimides-biotin (SH reactive), and carbodiimides-biotin (COOH reactive), biocytin hydrazide only requires a simple one-step chemical reaction without a solubility problem to biotinylated peptides/proteins. Fig. 4 shows the time course of biotinylating insulin and lysozyme using 1 M biocytin hydrazide in 10% (v/v) Fig. 2. The MALDI-TOF spectra of intact and biotinylated proteins (cytochrome c, lysozyme, trypsin inhibitor, and BSA): (I a) Cytochrome c at 37 C for 96 h in 10% TFA (v/v) aqueous solution without biocytin hydrazide; (I b) Cytochrome c at 37 C for 96 h in 10% TFA (v/v) aqueous solution with 0.3 M biocytin hydrazide; (II a) Lysozyme at 65 C for 24 h in 10% TFA (v/v) aqueous solution without biocytin hydrazide; (II b) Lysozyme at 65 C for 24 h in 10% TFA (v/v) aqueous solution with 1 M biocytin hydrazide; (III a) Trypsin inhibitor at 65 C for 19 h in PBS without biocytin hydrazide; (III b) Trypsin inhibitor at 65 C for 19 h in PBS with 1 M biocytin hydrazide; (IV a) BSA at 65 C for 19 h in PBS without biocytin hydrazide; (IV b) BSA at 65 C for 19 h in PBS with 1 M biocytin hydrazide. Protein solution (1 g/l) was processed with Zip-Tip packed with C4 resin before MALDI-TOF MS. Sinapinic acid was used as the matrix. Contaminant is marked by an asterisk. TFA at 65 C. Within 4 h of incubation, up to two biocytin hydrazide molecules are attached to both insulin and lysozyme. With longer incubation time, more biocytin hydrazide molecules attach to both insulin and lysozyme. After 24 h of incubation, up to 6 biocytin hydrazide molecules attach to both insulin and lysozyme. This data indicates that Fig. 3. The MALDI-TOF spectra of the reaction of insulin with 0.3 M of biocytin HCl, phenyl hydrazine, adipic dihydrazide, 2-iminobiotin, or 2iminobiotin hydrazide at 65 C and 5.5 h. Aqueous solution of 50% acetonitrile (v/v) containing 1% TFA (v/v) was used as the solvent. Peptide solution (1 g/l) was processed with Zip- Tip packed with C18 resin before MALDI-TOF MS. CHCA was used as the matrix. Fig. 4. Time courses of the biotinylation of insulin and lysozyme at 65 C with 1 M biocytin hydrazide in 10% TFA (v/v) aqueous solution by MALDI- TOF MS. Peptide/protein solution (1 g/l) was processed with Zip-Tip before MALDI- TOF MS.
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Biotinylation of Peptides/Proteins using Biocytin Hydrazide

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