ACTIN%20OLIGOPEPTIDES%20GENERATED%20DURING%20DRY-CURED%20HAM%20PROCESSING - PowerPoint PPT Presentation

Title: ACTIN%20OLIGOPEPTIDES%20GENERATED%20DURING%20DRY-CURED%20HAM%20PROCESSING


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ACTIN OLIGOPEPTIDES GENERATED DURING DRY-CURED
HAM PROCESSING
M.A.Sentandreu1, 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
RESULTS
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.
ABSTRACT
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.
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.
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 QKDSYVGDEA QSKRGILTLK YPIEHGIITN
WDDMEKIWHH TFYNELRVAP100 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 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.
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 (MH) at m/z 1501.5 and 1559.5 Da, respectively, in accordance with the peptide size stimated by size-exclusion chromatography.
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
Figure 2 MALDI-TOF MS of peak 1 obtained after
reverse-phase chromatography (Fig. 1). Black
arrows indicate ions that were further analyzed
by MS/MS.
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. ACKNOWLEDGEMENTS This work was
supported by an I3P contract from the European
Social Fund (to M.A.S.) a Marie Curie grant
(MERG-CT-2004-510652) from European Commission
(to M.A.S.) and by grant BFU-2004-01432 from the
Ministerio de Educación y Ciencia (Spain) (to
J.J.C.).
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 MH 1501.5)) and 780.8 (MH 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.