ACTIN OLIGOPEPTIDES GENERATED DURING DRY-CURED HAM PROCESSING
M.A.Sentandreu*1, M. Armenteros1, J.J. Calvete2, M.C. Aristoy1 and Fidel Toldrá1
1:
Instituto de Agroquímica y Tecnología de Alimentos (CSIC), P.O. Box 73, 46100 Burjassot, Valencia, Spain
2:
Instituto de Biomedicina de Valencia (CSIC). Jaime Roig 11, 46010. Valencia, Spain
Keywords: Dry-cured ham; proteolysis; actin; peptide sequencing; proteomics
2+
MS/MS 1501.5
ABSTRACT
PeptideA
RESULTS
DSGDGVTHNVPIYE
During the processing of dry-cured ham, there is an intense
proteolysis of muscle proteins mainly due to the action of
endogenous proteolytic enzymes. This gives rise to an
important generation of free amino acids and peptides of small
size, which contribute directly or indirectly to flavour
characteristics of the final product. The nature and properties of
free amino acids generated during postmortem proteolysis has
been well established by scientists but, on the contrary, little is
known about the identity of dry-cured ham peptide fraction at
the end of processing. In the present contribution we describe
the
isolation
and
identification
of
two
peptides,
DSGDGVTHNVPIYE and DSGDGVTHNVPIYEG, from this
fraction. Sequence homology analysis revealed that both
corresponded to the same region of muscle actin, differing only
in the presence or absence of Gly170 at the C-terminal position,
a fact that could be due to carboxypeptidase activity during the
curing period. Results of the present work show for the first time
the identification of specific actin fragments generated during
the processing of dry-cured ham.
Dry-cured ham extract was fractionated on a Sephadex G-25
column, pooling fractions eluting at 215-230 ml elution volume
which, according to a calibration curve from 180 to 12400 Da,
corresponded to a molecular mass of around 1530 Da.
Pooled fractions were concentrated and subjected to
preparative C18 reverse-phase chromatography. One
fraction, exhibiting a relevant absorbance value at =214 nm
and eluting at a concentration of 14 % acetonitrile, was
selected for the isolation and identification of peptides. For
this purpose, the selected fraction was subsequently
chromatographed on an analytical C18 reverse-phase
column, generating the chromatogram shown in Fig. 1.
MS/MS 1559.5
2+
Peptide B
DSGDGVTHNVPIYEG
Peak 1
Figure 3: CID MS/MS spectra of ions 751.92+ (Peptide A)
and 780.8 2+ (Peptide B) identified in peak 1 (Fig. 1).
Peptide sequences matching each of the product ion
spectra are shown in capital letters.
INTRODUCTION
Dry-cured ham is a traditional food requiring a long
processing period for development of its appreciated texture
and flavour characteristics. During this time, there is an
intense degradation of muscle proteins due to the action of
endogenous proteolytic enzymes. This gives rise to an
important generation of free amino acids and oligopeptides
that contribute directly or indirectly to flavour characteristics of
the final product. The nature and properties of free amino
acids has been well established but, on the contrary, little is
known about the identity of the peptide fraction at the end of
curing. The aim of the present work was to advance in the
knowledge of postmortem muscle proteolysis as related to the
quality of meat and meat products by implementing proteomic
technology, to overcome some of the difficulties found in the
past by meat scientist in this field.
METHODS
The methodology carried out for the isolation and identification of
peptides contained in dry-cured ham is the following:
BLAST sequence similarity searches revealed a 100 % identity
of these two peptides with an internal region of porcine muscle
actin, as can be observed in Fig. 4:
MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG50
Figure 1: Analytical C18 reverse-phase chromatography
of a selected fraction obtained after size-exclusion
chromatography
and
preparative
reverse-phase
chromatography of a dry-cured ham extract. Peak named
as “Peak 1” was further characterized by mass
spectrometry.
Fractions collected after this chromatography were further
analysed by MALDI-TOF MS. Peak 1 (Fig. 1) yielded a mass
spectrum showing molecular ions of appreciable intensity, as
can be observed in Fig. 2. MALDI-TOF MS of this fraction
yielded two quasimolecular ions (M+H+) at m/z 1501.5 and
1559.5 Da, respectively, in accordance with the peptide size
stimated by size-exclusion chromatography.
90
Dry-cured ham
80
% Intensity
70
60
50
40
Precipitated proteins
(Discarded)
Supernatant
- Redissolution in 0.01 N HCL
Size-exclusion chromatography
Preparative reverse-phase chromatography
Analytical reverse-phase chromatography
- Drying
- Redissolution in 50 % ACN-0.1 % TFA
MALDI-TOF
MS
30
20
- Drying
CID-MS/MS
EEHPTLLTEA PLNPKANREK MTQIMFETFN VPAMYVAIQA VLSLYASGRT150
TGIVLDSGDG VTHNVPIYEG YALPHAIMRL DLAGRDLTDY LMKILTERGY200
-----DSGDG VTHNVPIYE- (Peptide A)
-----DSGDG VTHNVPIYEG (Peptide B)
SFVTTAEREI VRDIKEKLCY VALDFENEMA TAASSSSLEK SYELPDGQVI250
TIGNERFRCP ETLFQPSFIG MESAGIHETT YNSIMKCDID IRKDLYANNV300
100
- Deproteinisation
QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWHH TFYNELRVAP100
10
0
469.0
716.6
964.2
1211.8
1459.4
1707.0
Mass (m/z)
Figure 2: MALDI-TOF MS of peak 1 obtained after reversephase chromatography (Fig. 1). Black arrows indicate
ions that were further analyzed by MS/MS.
These two peptide ions were subjected to ESI-ion trap in order
to elucidate their sequence. Collission-induced dissociation
MS/MS spectra of the doubly charged ions 751.9
(corresponding to the M+H+ 1501.5)) and 780.8 (M+H+ 1559.5),
shown in figure 3, matched the peptide sequence
DSGDGVTHNVPIYE (peptide A) and DSGDGVTHNVPIYEG
(peptide B), respectively. These two peptides share a common
sequence except for the C-terminal Glycine, present in peptide
B but absent from peptide A.
MSGGTTMYPG IADRMQKEIT ALAPSTMKIK IIAPPERKYS VWIGGSILAS350
LSTFQQMWIT KQEYDEAGPS IVHRKCF377
Figure 4: Primary structure of porcine skeletal muscle actin,
indicating the position of peptides A and B isolated and
identified in the present work.
The present results constitute a clear evidence of the intense
actin proteolysis occurring during dry-cured ham processing.
Previous works reported the progressive actin degradation
during postmortem muscle proteolysis either during meat ageing
but specially during dry-curing where most myofibrillar proteins
are extensively degraded. Other authors reported a notably
increase of the peptide fraction during dry-cured ham
processing, indicative of an intense proteolysis that is closely
related to flavour development. However, this is the first time
that small peptides coming from actin are isolated and fully
identified from dry-cured ham. The fact to identify two fragments
with and without Gly170 in C-terminal position may be indicative
of any exopeptidase action during the curing period. According
to previous observations, degradation of actin and other
myofibrillar proteins during postmortem muscle proteolysis could
be due to the action of lysosomal cathepsins, a hypothesis that
would be supported by their relatively good stability observed
during great part of the curing period
CONCLUSIONS
Two actin fragments of small size have (1501.5 and
1559.5 Da) been isolated and identified for the first
time in a dry-cured ham extract at the end of
processing, confirming the extensive proteolysis of
this protein during curing. These findings contribute to
know more about the complex mechanisms taking
place in postmortem muscle and whose enzyme
groups would be mainly implicated in the proteolytic
processes.
Peptide sequence
Molecular mass
ACKNOWLEDGEMENTS
BLAST sequence similarity searches
Protein origin
This work was supported by an I3P contract from the European
Social Fund (to M.A.S.) a Marie Curie grant (MERG-CT-2004510652) from European Commission (to M.A.S.) and by grant BFU2004-01432 from the Ministerio de Educación y Ciencia (Spain) (to
J.J.C.).