Biotinylation of Peptides/Proteins using Biocytin Hydrazide

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542 J. Chin. Chem. Soc., Vol. 54, No. 2, 2007 Lee et al. tide/protein occurs with the hydrazide moiety but not with the biotin moiety. This study shows that biocytin hydrazide can react readily with peptides/proteins through the amino and/or guanidino groups. Caution should be exercised when biotinylating the carbohydrate moiety of iodate-oxidized glycoproteins using biocytin hydrazide to alleviate this side reaction. EXPERIMENTAL SECTION Reagents All chemicals were purchased from Sigma (Saint Louis, MO) except EZ-Link TM Biocytin Hydrazide which was purchased from Pierce (Rockford, IL). The peptides and proteins with the highest purity grade were purchased from Sigma and used without further purification. Reactions between biocytin hydrazide and peptides/proteins Ten l of the peptide/protein solution (1 g/l) containing 0.01 to 1 M biocytin hydrazide was incubated at 4 C, 37 Cor65C for up to 4 days. PBS (0.01 M phosphate; 0.318 M NaCl; 0.027 M KCl; pH 7.4), 50 mM Na2CO3 aqueous solution at pH 10, or 10% (v/v) aqueous TFA (pKa 0.5) solution was used as the solvent. Ten l of the peptide/protein solution (1 g/l) at the conditions described above without biocytin hydrazide was incubated for the same time periods and used as a control. For Mass Spectrometric analysis, one l aliquots were removed at designated periods and diluted 10-fold with 0.1% TFA aqueous solution. Reactions of insulin with biocytin, 2-iminobiotin, and hydrazide derivatives Ten l of the insulin solution (1 g/l) containing 0.3 M biocytin HCl, phenyl hydrazine, adipic dihydrazide, 2-iminobiotin, or 2-iminobiotin hydrazide were incubated at 65 C for 5.5 h. An aqueous solution of 50% acetonitrile containing 1% (v/v) TFA was used as the solvent. Note that this solvent was selected so that all the materials can form a clear solution. Ten l of insulin solution (1 g/l) without the biotin derivative or hydrazide were incubated at the conditions described above and used as a control. For mass spectrometric analysis, one l aliquot was removed at designated time periods and diluted 10-fold with 0.1% (v/v) aqueous TFA solution. MALDI-TOF mass spectrometric analysis Zip-Tips (Millipore, Billerica, MA) packed with C18 and C4 resin were used to prepare the solution for MS analysis of peptides and proteins, respectively. -Cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid (SA) were used as the matrix for peptides and proteins, respectively. Aliquots (1.3 l) of the matrix solution [3-10 mg CHCA or SA in 1 mL aqueous solution of 50% (v/v) acetonitrile containing 0.1% (v/v) TFA] were used to elute the peptides or proteins from the ZipTips and spotted onto a MALDI-TOF target. A Voyager-DE PRO Mass Spectrometer (Applied Biosystems) equipped with a 337 nm pulsed nitrogen laser was used to analyze the samples. Masses of peptides or proteins were determined using the positive-ion linear mode. External mass calibration was performed using the peaks of a mixture of bradykinin fragments 1-7 at m/z 758, angiotensin II (human) at m/z 1047, P14R (synthetic peptide) at m/z 1534, adrenocorticotropic hormone fragment 18-39 (human) at m/z 2467, insulin oxidized B (bovine) at m/z 3497, insulin (bovine) at m/z 5735, cytochrome c (equine) at m/z 12362, apomyoglobin (equine) at m/z 16952, adolase (rabbit muscle) at m/z 39212, and albumin (bovine serum) at m/z 66430. The machine is calibrated for peptides and proteins separately. The biotinylated P14R atm/z 1904 was selected as a precursor ion using an ion gate set at a resolving power of 100 for positive MALDI-Postsource Decay (PSD) TOF analysis. PSD was produced using 15 kV accelerating potential with different reflector voltage segments. A composite mass spectrum, which contained the fragment ions for obtaining the sequence information, was produced by stitching 15 different mass spectra. Each spectrum was an average of 100 laser pulses containing a portion of the entire mass range. RESULTS AND DISCUSSION The molecular ions of human angiotensin I, P14R, human adrenocorticotropic hormone (ACTH) fragments 1- 13, and bovine insulin as well as their biotinylated ions are exhibited in Fig. 1. These peptides gain 370 Da upon condensation with one biocytin hydrazide molecule. Molecular masses of these biotinylated peptides produced by incubating peptides with biocytin hydrazide and the number of biocytin hydrazide molecules attached to the biotinylated peptides are compiled in Table 1. The biotinylated ions
Biotinylation of Peptides/Proteins using Biocytin Hydrazide J. Chin. Chem. Soc., Vol. 54, No. 2, 2007 543 were detected at m/z 1667 (singly biotinylated) in the case of agiotensin I, at m/z 1904 (singly biotinylated) in the case of P14R, at m/z 1994 (singly biotinylated) in the case of ACTH fragments 1-13, and at m/z 6105 (singly biotinylated), m/z 6475 (doubly biotinylated), m/z 6845 (triply biotinylated), m/z 7215 (quadruply biotinylated), m/z 7585 (five biotinylations), m/z 7855 (six biotinylations), and at m/z 8325 (seven biotinylations) in the case of insulin. Since there is only one biotinylation detected in the case of angiotensin I and the sequence of angiotensin I (DRVYIHPFHL) contains no lysine residue, N-terminal or other animo acid residue besides lysine is biotinylated. Results from a MALDI-TOF post-source decay (PSD) study of m/z 1904 (singly biotinylated P14R) described later together with that only one biotinylation was detected in the case of P14R Fig. 1. The MALDI-TOF spectra of intact and biotinylated peptides (angiotensin, P14R, ACTH fragment 1-13, and insulin) at 65 C for 24 h: (I a) Angiotensin I in 10% TFA (v/v) aqueous solution without biocytin hydrazide; (I b) Angiotensin I in 10% TFA (v/v) aqueous solution with 1 M biocytin hydrazide; (II a) P14R in 10% TFA (v/v) aqueous solution without biocytin hydrazide; (II b) P14R in 10% TFA (v/v) aqueous solution with 1 M biocytin hydrazide; (III a) ACTH fragment 1-13 in 10% TFA (v/v) aqueous solution without biocytin hydrazide; (III b) ACTH fragment 1-13 in 10% TFA (v/v) aqueous solution with 1 M biocytin hydrazide; (IV a) Insulin in PBS without biocytin hydrazide; (IV b) Insulin in PBS with 1 M biocytin hydrazide. Peptide solution (1 g/l) was processed with Zip-Tip packed with C18 resin before MALDI- TOF MS. CHCA was used as the matrix. Contaminant is marked by an asterisk. (PPPPPPPPPPPPPPR) indicate that it is the arginine residue of P14R which is biotinylated. In addition, PSD results (data not shown) of ACTH and angiotensin I show that the arginine side chain for both peptides is biotinylated. Since there are seven biotinylations detected in the case of insulin and the sequence of insulin (GIVEQCCASVCSLYQLEN- YCNFVNQHLCGSHLVEALYLVCGERGFFYTPKA) contains only one lysine, one arginine and two N-terminals, it seems that residues other than lysine or arginine can also react with biocytin hydrazide. It is noted that unlike the bisulfite-catalyzed (rate increases up to 100 times) transamination reaction of cytidine residues in DNA with biotin hydrazide, 2,7-11 addition of sodium bisulfite (1 M) only slightly increases the reaction rate of peptides/proteins with biocytin hydrazide (data not shown). The molecular ions of horse heart cytochrome c, hen egg white lysozyme, soybean trypsin inhibitor (SBTI), and bovine serum albumin (BSA) as well as their biotinylated ions are exhibited in Fig. 2. These proteins gain 370 Da upon condensation with one biocytin hydrazide molecule. Molecular masses of these biotinylated proteins produced by incubating proteins with biocytin hydrazide and the number of biocytin hydrazide molecules attached to the proteins are compiled in Table 1. The biotinylated ions were detected at m/z 12732, 13102, 13472, 13842, 14212, and 14582 (up to 6 biotinylations) in the case of cytochrome c, at m/z 14676, 15046, 15416, 15786, 16156, and 16526 (up to 6 biotinylations) in the case of lysozyme, at m/z 20365, 20735, 21105, and 21475 (up to 4 biotinylations) in the case of SBTI, and m/z 72720 (approximately 17 biotinylations) and m/z 83450 (approximately 46 biotinylations) in the case of BSA. In general, since there are mixtures of many biotinylated proteins with different biocytin hydrazide molecules attached, the peaks corresponding to biotinylated proteins are broader than their nonbiotinylated counterparts. Since there are more lysine residues than the total number of biotinylations of each protein, not all the lysine residues in each protein are reactive toward biocytin hydrazide. The MALDI-TOF spectra of the products produced by reacting insulin with 0.3 M of biocytin HCl, phenyl hydrazine, adipic dihydrazide, 2-iminobiotin, or 2-iminobiotin hydrazide at 65 C for 5.5 h are shown in Fig. 3, and masses of the various biotinylated insulins are compiled in Table 1. An aqueous solution of 50% (v/v) acetonitrile containing 1% (v/v) TFA was used as the solvent to solubilize all the materials. Insulin should gain 355, 241, 226, 157,
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Biotinylation of Peptides/Proteins using Biocytin Hydrazide

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