Biotinylation of Peptides/Proteins using Biocytin Hydrazide

Biotinylation of Peptides/Proteins using Biocytin Hydrazide 

Bao-Shiang Lee,* Sangeeth Krishnanchettiar, Syed Salman Lateef and Shalini Gupta 

Protein Research Laboratory, Research Resources Center, University of Illinois, 835 S. Wolcott Avenue, 

Chicago, Illinois 60612, USA 

Biocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In this 

study, however, biocytin hydrazide was found to be able to directly biotinylate peptides and proteins. This 

phenomenon may cause false identification of non-glycopeptides/non-glycoproteins as glycopeptides/ 

glycoproteins. Here, we report a systematic investigation of the reaction of peptides/proteins with biocytin 

hydrazide. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is used to analyze 

the biotinylation reaction between peptides/proteins and biocytin hydrazide. Peptides/proteins were reacted 

with biocytin hydrazide in diverse solvent systems with different biocytin hydrazide concentrations 

for up to 96 h at temperatures ranging from 4 Cto65C. Singly biotinylated or multiply biotinylated peptides/proteins 

are observed. The efficiency of the biotinylation reaction increases with higher temperature, 

higher biocytin hydrazide concentration, or longer reaction time. The influence of buffer pH on the 

biotinylation reaction of peptides/proteins is less pronounced. The biotinylation efficiency is optimum at 

neutral pH. Data suggests that the peptides are biotinylated as efficiently as proteins. The observation that 

peptides/proteins condense only with biocytin hydrazide, 2-iminobiotin hydrazide, adipic dihydrazide 

and phenyl hydrazine but not with biocytin HCl and 2-iminobiotin, indicates that the biotinylation reaction 

of peptides/proteins occurs with the hydrazide moiety but not with biotin moiety of the biotinylated 

reagent. The postsource decay data of biotinylated P14R indicates that biocytin hydrazide condenses with 

the guanidino group of arginine’s side chain of P14R, indicating that besides N-terminal and lysine residue 

of peptides/proteins, arginine residue is capable of reacting with biocytin hydrazide. 

Keywords: Biocytin hydrazide; Peptides; Proteins; Biotinylation; MALDI-TOF MS. 

INTRODUCTION 

The biotin-avidin system is widely used in many biochemical 

techniques 1 for its strong non-covalent biotinavidin 

bonding with a dissociation constant of ~10 -15 M. 

One of such techniques uses biocytin hydrazide to label oxidized 

glycoproteins. 2 The process involves oxidation of 

the glycoproteins by iodate to convert the hydroxyl residues 

of the sugar into reactive aldehyde. After that, a hydrazone 

bond was formed by reacting biocytin hydrazide with 

aldehyde. Subsequently, biotinylated protein is captured 

and analyzed by bioanalytical techniques. 2-6 However, the 

non-specific reactions between peptides/proteins and biocytin 

hydrazide remain to be understood. The non-specific 

reactions occur through the interaction of amino groups of 

peptides/proteins with the hydrazide group of the biocytin 

hydrazide, presumably similar to the transamination reaction 

of biotin hydrazide with cytidine residues in DNA. 2,7-11 

Journal of the Chinese Chemical Society, 2007, 54, 541-548 541 

* Corresponding author. Tel: (312) 996-1411; Fax: (312) 996-1898; E-mail: boblee@uic.edu 

In this study, we conduct a study on biotinylation of peptides/proteins 

with biocytin hydrazide using the simple, 

fast, and informative Matrix-assisted laser desorption/ionization 

time-of-flight mass spectrometry (MALDI-TOF 

MS). 12 Human angiotensin I (DRVYIHPFHL, m/z 1297), 

P14R (PPPPPPPPPPPPPPR, m/z 1534), human adrenocorticotropic 

hormone fragment 1-13 (SYSMEHFRWGKPV, 

m/z 1624), bovine insulin (m/z 5735), cytochrome c (m/z 

12362), lysozyme (m/z 14,300), trypsin inhibitor (m/z 

20,000), and BSA (m/z 66,430) were used as test cases. The 

effects of reaction time, biocytin hydrazide concentration, 

buffer pH, or temperature on the peptides/proteins’ biotinylation 

were also investigated. In addition, besides biocytin 

hydrazide, 2-iminobiotin hydrazide, adipic dihydrazide, 

phenyl hydrazine, biocytin HCl, and 2-iminobiotin 

were also tested as biotinylation reagents. Data suggests 

that peptides/proteins can be efficiently biotinylated with 

biocytin hydrzide, and the biotinylation reaction of pep-

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,


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.


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.


the efficiency of biotinylation of peptides and proteins increases 

with longer reaction time. Further increase in incubation 

time for both insulin and lysozyme produces lesser 

effect on the efficiency of biotinylation. This may indicate 

that the reaction may have reached a saturation state. However, 

an increase in the degradation products was observed 

upon prolonged incubation for all the peptides/proteins 

studied here. 

Fig. 5 shows the MALDI-TOF mass spectra of the 

biotinylated products of insulin and BSA as a function of 

the biocytin hydrazide concentration. Several different biocytin 

hydrazide concentrations (0.01 M, 0.1 M,or1M) are 

used to biotinylate insulin in 10% (v/v) TFA and to biotinylated 

BSA in PBS at 65 C for 5 h. At 0.01 M of biocytin 

hydrazide there are an average of 1.4 biotinylations in BSA 

but there is almost no observable biotinylation in insulin. 

At 0.1 M of biocytin hydrazide there are an average of 1.7 

biotinylations in BSA and one biotinylation in insulin. At 1 

M of biocytin hydrazide there are an average of 17 biotinylations 

in BSA and up to 2 biotinylations in insulin. This 

data indicates that biotinylation efficiency of proteins increases 

with biocytin hydrazide concentrations, though the 

progression is not linear. 

Fig. 6 shows the effect of temperature on biotinylation 

reaction of insulin in 10% (v/v) TFA aqueous solution 

and on biotinylation reaction of BSA in PBS using 1 M 

biocytin hydrazide. The reaction times are 5 h for both insulin 

and BSA. At 4 C, an average of 6.4 biotinylations are 

Fig. 5. The biotinylation of insulin and BSA at 65 C 

and5hin10%TFA(v/v) aqueous solution with 

biocytin hydrazide as a function of biocytin 

hydrazide concentration and analyzed by 

MALDI-TOF MS. Peptide/protein solution (1 

g/l) was processed with Zip-Tip before 

MALDI-TOF MS. 

observed in BSA but none in insulin. At 37 C, an average 

of 8.6 biotinylations in BSA and one biotinylation in insulin 

are observed. At 65 C, an average of 17 biotinylations 

in BSA and up to five biotinylations in insulin are observed. 

Data shows that higher temperatures speed up the 

biotinylation reaction in a non-linear fashion. 

Fig. 7 shows the effect of pH on the biotinylation of 

Fig. 6. The biotinylation of insulin in 10% TFA (v/v) 

aqueous solution and BSA in PBS at 65 C with 

1 M biocytin hydrazide as a function of temperature 

and analyzed by MALDI-TOF MS. The 

incubation time was 24 h and 5 h for insulin and 

BSA, respectively. Peptide/protein solution (1 

g/l) was processed with Zip-Tip before 

MALDI-TOF MS. 

Fig. 7. Buffer pH dependence of the biotinylation of 

insulin (65 C, 24 h) and BSA (65 C, 5 h) with 

1 M biocytin hydrazide by MALDI-TOF MS. 

Peptide/protein solution (1 g/l) was processed 

with Zip-Tip before MALDI-TOF MS.


insulin and BSA using 1 M biocytin hydrazide at 65 C. It is 

noted that the influence of pH on the biotinylation efficiency 

of peptides/proteins is less pronounced. The reaction 

times were 24 h and 5 h for insulin and BSA, respectively. 

With the 10% (v/v) aqueous TFA (pKa 0.5) as the 

solvent, up to 12 biotinylations for BSA and up to five 

biotinylations for insulin are observed. With PBS (pH 7.4) 

as the solvent, an average of 17 biotinylations for BSA and 

up to seven biotinylations for insulin are observed. With 50 

mM Na2CO3 (pH 10) as the solvent, there are an average of 

6.6 biotinylations for BSA and up to four biotinylations for 

insulin. Data shows that neutral pH is perhaps the optimum 

for biotinylation of peptides/proteins using biocytin hydrazide. 

Fig. 8 shows the MALDI-PSD TOF spectrum of biotinylated 

P14Ratm/z 1904. Biotinylated P14R was produced 

by reacting with 1 M biocytin hydrazide at 65 C for 24 h. 

10% TFA aqueous solution (v/v) was used as the solvent. 

Data displays “y” (bond-cleavage fragments that retain 

C-terminal side of the peptide) fragment ions. The detected 

y11 (m/z 1519), y12 (m/z 1613), y13 (m/z 1710), and y14 (m/z 

1807) fragment ions are in agreement with the m/z values of 

the fragments carrying a biocytin hydrazide, indicating that 

the biocytin hydrazide is attached to the arginine side chain 

of the peptide because if biocytin hydrazide is attached to 

the proline residue, one would observe many multiply biotinylated 

P14R. It is noted that PSD results (data not shown) 

of ACTH and angiotensin I show that the arginine side 

chain for both peptides is biotinylated. 

Fig. 8. The MALDI-PSD TOF spectrum of biotinylated 

P14R. Biotinylated P14R was produced by 

reacting with 1 M biocytin hydrazide at 65 C 

for 24 h. 10% TFA (v/v) aqueous solution was 

used as the solvent. Peptide solution (1 g/l) 

was processed with Zip-Tip before MALDI- 

TOF MS. 

CONCLUSIONS 

MALDI-TOF Mass Spectrometry has been successfully 

used to investigate the biotinylation of peptides/proteins 

using biocytin hydrazide. Besides the physiological 

conditions, harsh conditions (65 C; higher concentration 

of biocytin hydrazide; higher and lower pH; longer reaction 

time) are also used in this study. Results indicate that biotinylation 

of peptides/proteins is readily accomplished with 

biocytin hydrazide. The efficiency of the biotinylation reaction 

increases with higher temperature, higher biocytin 

hydrazide concentration, or longer reaction time. The influence 

of buffer pH on the biotinylation reaction of peptides/ 

proteins is less pronounced. The biotinylation efficiency is 

optimum at neutral pH. Post-source decay data of biotinylated 

P14R, ACTH, and angiotensin I indicate that biocytin 

hydrazide reacts with the guanidino group present on the 

side chain of the arginine residue. Since the peptides/proteins 

react with biocytin hydrazide, 2-iminobiotin hydrazide, 

adipic dihydrazide and phenyl hydrazine but not with 

biocytin HCl and 2-iminobiotin, the biotinylation reaction 

of peptides/proteins should occur with the hydrazide moiety 

but not with biotin moiety of the biotinylated reagent. In 

short, 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. 

ACKNOWLEDGEMENTS 

We thank the Research Resources Center at the University 

of Illinois at Chicago for their support. 

Received April 10, 2006. 

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Biotinylation of Peptides/Proteins using Biocytin Hydrazide

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