Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis.
In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate comparison of 2D maps to define the events occurring during their ripening and individuate candidate molecular markers of the characteristic proteolytic processes. Considering that, essentially, muscle endogenous enzymic activity, calpains and cathepsins, occur in the ripening process of dry-cured ham, whereas a combined action between endogenous and microbial enzymes takes place in the case of sausage ripening, these results provide deeper insight into the respective role of endogenous and microbial enzymes in performing proteolysis. Finally, image analysis and creation of data bank could be achieved to quickly identify and protect typical products.
Picariello
Gianluca
G
Istituto di Scienze dell'Alimentazione-CNR, Via Roma 52A/C, 83100 Avellino, Italy.
De Martino
Alessandra
A
Mamone
Gianfranco
G
Ferranti
Pasquale
P
Addeo
Francesco
F
Faccia
Michele
M
Spagnamusso
Salvatore
S
Di Luccia
Aldo
A
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D009124:Muscle Proteins / Q000737:chemistry*; D040901:Proteomics; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D009124:Muscle Proteins