Actin oligopeptides generated during dry cured ham processing
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ACTIN OLIGOPEPTIDES GENERATED DURING DRY-CURED HAM PROCESSING PowerPoint PPT Presentation


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2+. MS/MS 1501.5. Peptide A. DSGDGVTHNVPIYE. MS/MS 1559.5. 2+. Peptide B. DSGDGVTHNVPIYEG. METHODS. The methodology carried out for the isolation and identification of peptides contained in dry-cured ham is the following:. 100. 90. Dry-cured ham. 80. 70. - Deproteinisation.

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ACTIN OLIGOPEPTIDES GENERATED DURING DRY-CURED HAM PROCESSING

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Actin oligopeptides generated during dry cured ham processing

2+

MS/MS 1501.5

PeptideA

DSGDGVTHNVPIYE

MS/MS 1559.5

2+

Peptide B

DSGDGVTHNVPIYEG

METHODS

The methodology carried out for the isolation and identification of peptides contained in dry-cured ham is the following:

100

90

Dry-cured ham

80

70

- Deproteinisation

60

% Intensity

50

40

Precipitated proteins

(Discarded)

Supernatant

30

20

- Drying

- Redissolution in 0.01 N HCL

10

0

Size-exclusion chromatography

469.0

716.6

964.2

1211.8

1459.4

1707.0

Mass (m/z)

Preparative reverse-phase chromatography

Analytical reverse-phase chromatography

- Drying

- Redissolution in 50 % ACN-0.1 % TFA

MALDI-TOF MS

CID-MS/MS

Peptide sequence

Molecular mass

BLAST sequence similarity searches

Protein origin

M.A.Sentandreu*1, M. Armenteros1, J.J. Calvete2, M.C. Aristoy1and 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

ACTIN OLIGOPEPTIDES GENERATED DURING DRY-CURED HAM PROCESSING

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.

MCDEDETTAL VCDNGSGLVK AGFAGDDAPRAVFPSIVGRPRHQGVMVGMG50

QKDSYVGDEAQSKRGILTLK YPIEHGIITN WDDMEKIWHHTFYNELRVAP100

EEHPTLLTEA PLNPKANREKMTQIMFETFN VPAMYVAIQA VLSLYASGRT150

TGIVLDSGDG VTHNVPIYEG YALPHAIMRLDLAGRDLTDY LMKILTERGY200

-----DSGDG VTHNVPIYE- (Peptide A)

-----DSGDG VTHNVPIYEG (Peptide B)

SFVTTAEREIVRDIKEKLCY VALDFENEMA TAASSSSLEKSYELPDGQVI250

TIGNERFRCP ETLFQPSFIGMESAGIHETT YNSIMKCDID IRKDLYANNV300

MSGGTTMYPG IADRMQKEIT ALAPSTMKIKIIAPPERKYS VWIGGSILAS350

LSTFQQMWITKQEYDEAGPS 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.

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.).


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