Biotinylation of Peptides/Proteins using Biocytin Hydrazide

Biotinylation of Peptides/Proteins using Biocytin Hydrazide J. Chin. Chem. Soc., Vol. 54, No. 2, 2007 547
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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