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10-15 charged amino acids at most,
located at the C terminus.
Taking into account the natural abun-
dance of basic amino acids, the latter condi-
tion is an expected result after the tryptic
digestion of proteins and peptides. However,
the inconvenience associated with the
determination of cyclotides by MS/MS is
related to the presence of the CCK, which
requires the previous breakage and alkyla-
tion of the disulfide bonds and the rupture
of its cyclic backbone. Due to the scarcity of
positively charged amino-acid residues, the
enzymatic degradation renders undesired
fragments that are too long or that have an
inadequate charge (Göransson
et al.
, 2003).
In order to obtain fragments with a suitable
size for analysis, in this work, the endopro-
tease Glu-C was employed along with
trypsin.
To verify the amino-acid sequence by
MS/MS, the cyclotide was reduced and
treated with iodoacetamide, generating a
series of derivatives with a mass increase of
58 Da per each reduced Cys that reacts with
iodoacetamide. The cleavage with Glu-C
rendered a linear peptide of 30 amino acids
in length (from S1 to E30). Because this lin-
ear peptide was too long to be analysed by
MS/MS it was treated with trypsin, obtain-
ing three tryptic fragments: S1 to K18, N19
to K20 and V21 to E30. These peptides were
isolated by RP-HPLC and analysed by nano-
spray ion trap MS/MS (35% CID). The
experimental masses [M+H]
+
were assigned
to the fragments according to Biemann's
nomenclature (Biemann, 1990). The two
sequenced peptides were 28-30 amino
acids long.
The assignment of the
b
series
(N-terminus fragments) and the
y
series
(C-terminus fragments) allowed the verifica-
tion of the tryptic fragment of 18 amino acids,
S1 to K18. It was observed that the pres-
ence of lysine (K), which is a basic residue,
confers a positive charge on the fragment.
In the case of the 10 amino acid long
tryptic fragment (V21 to E30), the fragmen-
tation of the ion 1183.3 [M+H]
+
allowed
verification of the 10 amino acids located
before the cleavage site of Glu-C: V21 to E30
(Broussalis
et al.
, 2001).
The low frequency of basic amino acids
or, in this case, the complete absence of
them, and the formation of clusters of basic
amino acids, do not allow a precise assigna-
tion to this peptide. In this spectrum b
o
is
b- H
2
O. In this peptide the presence of lysine
(K) was not observed, whereas glutamic
acid (E) was present, an acidic residue that
confers a negative charge to this fragment.
The remnant peptide N19-K20 was con-
firmed by tandem MS of the linear peptide
of 30 amino acids obtained by partial diges-
tion with trypsin (Fig. 13.2).
This novel cyclotide exhibited a great
sequence homology with that of the cyclo-
tides previously identified in the Violaceae
and Rubiaceae families (Craik
et al
., 2001).
Furthermore, this cyclotide is 90% identi-
cal to the most closely related homologue,
cycloviolacin O1, which has been isolated
from
Viola odorata
. The disulfide bonds
between the Cys residues in hypa A,
C2-C17, C7-C22 and C15-C28 are identical
to those reported by Craik
et al
. (1999) for
cycloviolacin O1, as determined by nuclear
magnetic resonance (NMR).
According to the sequence alignment,
hypa A would belong to the subfamily 1,
that is, the 'bracelet' cyclotides, owing to the
absence of a
cis
-amide link between Trp2
and Pro3 (absence of a
cis
-Pro residue in the
loop 3), a feature of the subfamily 2, the
Möebius cyclotides (Craik
et al
., 2002). It is
also noteworthy that there is a difference in
the net charge between both families: as a
rule, the peptides belonging to the subfamily
1 have a net charge of +2, the value found for
hypa A, whereas those cyclotides belonging
Fig. 13.2.
The amino acid sequence of the cyclotide
hypa A (Broussalis
et al
., 2001). The cleavage site
of endoproteinase Glu-C, marked with an arrow,
was chosen as an arbitrary starting point of the
numbering of the amino acids (trypsin cleavage
sites are marked with dashed arrows).
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