Two-dimensional gel and shotgun proteomics approaches to distinguish fresh and frozen-thawed curled octopus (Eledone cirrhosa)

doi:10.1016/j.jprot.2018.07.017

Journal of Proteomics

Volume 186, 30 August 2018, Pages 1-7

https://doi.org/10.1016/j.jprot.2018.07.017 Get rights and content

Highlights

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Two proteomic approaches to differentiate the state of conservation are proposed.
•
Two 2-DE spots, characterized by MS/MS as transgelin, differentiate samples.
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Transgelin could be a marker to distinguish fresh and frozen-thawed curled octopus.

Abstract

The substitution and sale of frozen-thawed fish labeled as fresh is a widespread, difficult to unmask commercial fraud and a potential risk for consumer health. Proteomics could help to identify markers for the rapid screening of food samples and the identification of frozen-thawed seafood. Using two-dimensional electrophoresis (2-DE) and high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified biomarkers that are able to discriminate between fresh and frozen-thawed tissue samples of curled octopus (Eledone cirrhosa). The 2-DE analysis showed a significant reduction in two protein spots (molecular weight of 45–50 kDa, isoelectric point of 6.5–7) identified as transgelin. At shotgun analysis, nine proteins resulted modulated and transgelin was confirmed as down-regulated, making it a potentially useful marker for differentiating between fresh and frozen-thawed fish product samples.

Biological significance

This work, based on two different proteomics approaches, investigated differentially expressed proteins in the tentacles of the curled octopus (E. cirrhosa) after freezing-thawing processes. We were able to characterize the proteome of the tentacles, increasing our knowledge on this species, and a common down-regulated protein was identified by 2-DE and shotgun analysis, a calponin-like protein called transgelin, suggesting a potential use as a marker to distinguish different states of conservation in this species.

Graphical abstract

Introduction

With the increasing globalization of the food chain supply, finding ways to counteract food fraud has become essential to ensure food safety and quality. In the seafood sector, selling frozen-thawed fish labeled as fresh is a widespread practice that constitutes commercial fraud in violation of Regulation (EU) No. 1169/2011 on the provision of food information to consumers. It also poses a potential risk for consumer health because repeated freezing-thawing cycle can activate residing dormant micro flora and increase total microbial count.

Differentiating between fresh and frozen-thawed fish is not easy because, when proper freezing/thawing procedures are followed, the sensory properties are very similar to those of the fresh product [1]. Official inspection relies on the qualitative evaluation of muscle consistency, eye opacity, among other product characteristics. There are numerous alternative methods: measurement of electric resistance of muscle tissue [2], mitochondrial enzyme analysis [3], microbiological [4] and organoleptic [5] analysis, detection of changes in dielectric properties with nuclear magnetic resonance [6] and magnetic resonance imaging [7]. All require highly skilled operators and are relatively destructive and/or expensive and time-consuming. More recently, noninvasive and nondestructive techniques based on infrared spectroscopy and front-face fluorescence spectroscopy [[8], [9], [10]] now offer high performance results. Histopathological evaluation of specific microscopic changes related to freezing could be a valid method to differentiate fresh from frozen-thawed fish [11] but, like the methods mentioned above, it is not useful when applied to seafood products such as cephalopod molluscs.

The class Cephalopoda contains 21 families, each consisting of a large number of species, many of which of high commercial importance, include diverse species of octopus, squid, and cuttlefish. The curled octopus Eledone cirrhosa [12], the object of the present study for its commercial interest, is widely distributed over the shelf regions of the northeastern Atlantic and Mediterranean Sea [13].

Generally, because freezing and subsequent thawing cause protein denaturation, a proteomics method based on two-dimensional electrophoresis (2-DE) could provide a valid alternative approach to distinguish between fresh and frozen-thawed samples, as previously shown for sea bass (Dicentrarchus labrax) and cod (Gadus morhua) [14, 15]. Among currently available proteomic technologies, shotgun proteomics through the use of SWATH-MS (sequential windowed acquisition of all theoretical fragment ion mass spectra) [16] has been extensively employed to analyze complex biological samples and obtain accurate and reliable label-free quantification of proteins [17, 18]. The aim of this study was to find biomarkers that could help to distinguish fresh from frozen-thawed curled octopus using two-dimensional electrophoresis (2-DE) and shotgun proteomics (Fig. 1).

Section snippets

Samples

A total of 18 Eledone cirrhosa (curled octopus) were examined, and each sample was analyzed in triplicate. The analyses were performed on skinless tentacles divided in two aliquots. For each sample, the first aliquot, labeled “fresh”, was processed immediately to extract proteins, while the second was frozen at −20 °C for 72 h and thawed before protein extraction.

Protein extraction

Soluble proteins were obtained from 0.2–0.3 g of fresh or frozen–thawed skinless tentacles placed in 10 volumes (w/v) of buffer

2D-electrophoresis comparison of fresh and frozen-thawed samples

The 2-DE was used to compare protein maps of fresh versus frozen-thawed skinless tentacles of 18 samples of E. cirrhosa. Two representative 2-DE maps are shown in Fig. 2. Both images show about 280 spots distributed along the pH gradient. Focusing in particular on protein spots with isoelectric values of about 6.5–7 and molecular weight of about 45–50 kDa, the 2-DE profiles of 16 frozen-thawed samples showed a decrease in two protein spots as compared with the fresh samples. Approximately, in

Discussion

We applied two proteomic approaches to differentiate fresh from frozen-thawed curled octopus (E. cirrhosa). The 2-DE analysis revealed a quantitative variation of two protein spots that were decreased in the frozen-thawed samples as compared to the fresh sample. The two spots of interest identified in 2-DE were characterized by LC-MS/MS as transgelin, a protein belonging to the calponin family. Calponin-like proteins are actin-binding proteins that, together with caldesmon, act as a thin

Conclusion

In this study comparative analysis with 2-DE and shotgun proteomics are performed to differentiate fresh from frozen-thawed curled octopus (E. cirrhosa). The 2-DE revealed two protein spots that were decreased in the frozen-thawed samples; the LC-MS/MS analysis identified these two protein spots as transgelin. The shotgun proteomic approach and the label-free quantification confirmed the modulation of transgelin, suggesting that it has potential use as a marker to distinguish different states

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the Ministry of Health [Ricerca Corrente n° IZS PLV 01/14 RC]. The authors wish to thank Francesca Martucci for the supply of fresh E. cirrhosa samples.

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Two-dimensional gel and shotgun proteomics approaches to distinguish fresh and frozen-thawed curled octopus (Eledone cirrhosa)

Highlights

Abstract

Biological significance

Graphical abstract

Introduction

Section snippets

Samples

Protein extraction

2D-electrophoresis comparison of fresh and frozen-thawed samples

Discussion

Conclusion

Conflicts of interest

Acknowledgements

Food Res. Int.

Magn. Reson. Imaging

J. Food Prot.

Food Chem.

Biochim. Biophys. Acta Bioenerg.

Anal. Biochem.

J. Proteome

J. Biochem. Biotech.

Comparison of methods of differentiating between fresh and frozen-thawed fish or fillets

J. Sci. Food Agric.

Use of β-hydroxyacyl-CoA-dehydrogenase (HADH) activity to differentiate frozen from unfrozen fish and shellfish

Eur. Food Res. Technol.

Shelf life of thawed cod fillets kept in ice

Z. Lebensm. Unters. Forsch.

Consumer acceptability of cod and whiting after chilled storage and freezing and thawing

Int. J. Food Sci. Technol.

High-Resolution NMR and Magnetic Resonance Imaging (MRI) Studies on Fresh and Frozen Cod (Gadus morhua) and Haddock (Melanogrammus)

J. Sci. Food Agric.