6B Peptides 199
masses ranging from 16900 to 4400Da in a model wine during yeast autolysis. The
authors used the Sch ̈agger and von Jagow’s method (Sch ̈agger and von Jagow 1987)
for discontinuous electrophoresis and silver staining.
Desportes et al. (2000, 2001) used capillary electrophoresis on fused-silica capil-
laries with 25 mM, pH 2.5 phosphoric acid as running buffer, to verify the purity of
collected peaks after separation by HPLC. Both methods and some others described
for the analysis of wine proteins, such as that described by Luguera et al. (1997),
with slight modifications, could also be used for the analysis of medium molecular
weight peptides.
6B.2.5 Detection Methods
Peptides are commonly detected by absorbance at 200–220 nm. However, most of
the compounds present in wine may interfere in the ultraviolet detection of peptides
when low wavelengths are used. Thus, for the analysis of these compounds it is
useful to apply sensitive and selective detection methods. To this end, it is possible
to form derivates of the peptides that can be detected at higher and more specific
wavelengths. Detection by fluorescence can also be used to detect peptides contain-
ing fluorescence amino acids (tyrosine and tryptophan). For peptides without this
property, the formation of derivates with derivatizing agents have been proved to be
very useful (Moreno-Arribas et al. 1998a).
It is possible to use the formation of both pre- and post-column derivates. Post-
column reaction with OPA has the advantagethat enables the detection of natural
peptides by ultraviolet and in a second step the on-line detection of the derivates
by fluorescence). Bartolom ́e et al. (1997) reported the use of an on-line HPLC-
photodiode array detection (HPLC-PDAD) and a post-column OPA derivatization
system to characterize small peptidesfrom wine. This methodology was initially
applied to a wide range of standard peptides and, in further studies, it was suc-
cessfully applied to identify the peptide fraction (<700 Da) of sparkling wines.
Spectral parameters, such as wavelengthsof the spectrum peaks, convexity inter-
val, and wavelengths of the second-derivate spectrum peaks, obtained by PDAD,
allow the identification of the aromatic amino acid residues within the peptides and
also the identification of other compounds in wine that can coelute with peptides
(i.e. phenolic compounds). Figure 6B.3 shows the original and the first and second
derivative spectra of several peptides and Fig. 6B.4 the flowchart for the interpre-
tation of both spectral data and OPA-fluorescence response proposed by Moreno-
Arribas et al. (1998a) for the identification of peptides.
LC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass
spectrometry) has been used by Person et al. (2004) for identification and quan-
tification of di- and tripeptides in champagne wine after filtration with a Mr 10,000
cut-off membrane. This procedure avoids the time-consuming prepurification and
isolation steps.