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.).