Top-down HPLC-ESI-MS characterization of rat gliadoralin A, a new member of the family of rat submandibular gland glx-rich proteins and potential substrate of transglutaminase.

Massimo Cordaro, Massimo Castagnola, Tiziana Cabras, Maria Cristina De Rosa, Alberto Vitali, Gavino Faa, Irene Messana, Jörgen Ekström

Risultato della ricerca: Contributo in rivistaArticolo in rivista

3 Citazioni (Scopus)

Abstract

During HPLC-ESI-MS/MS analysis of rat submandibular saliva secreted under isoprenaline stimulation, a protein with an experimental [M+H](1+) = 10,544.24 m/z was detected (17.5 ± 0.7 min). The MS/MS fragmentation pattern, manually investigated, allowed establishing an internal sequence in agreement with a DNA-derived sequence of an unknown rat protein coded D3Z9M3 (Swiss-Prot). To match the experimental MS/MS fragmentation pattern and protein mass with theoretical data, the removal from the N terminus of the signal peptide and from the C terminus of three amino acid (a.a.) residues (Arg-Ala-Val) and the cyclization of the N-terminal glutamine in pyroglutamic had to be supposed, resulting in a mature protein of 90 a.a. HPLC-ESI-MS/MS of the trypsin digest ensured 100% sequence coverage. For the high glutamine content (34/90 = 37.8%) we propose to name this protein rat gliadoralin A 1-90. Low amounts of five different isoforms were sporadically detected, which did not significantly change their relative amounts after stimulation. Gliadoralin A is substrate for transglutaminase-2, having Lys 60 and different Gln residues as major determinants for enzyme recognition. In silico investigation of superior structures evidenced that a small part of the protein adopts an α-helical fold, whereas large segments are unfolded, suggesting an unordered conformation.
Lingua originaleEnglish
pagine (da-a)2848-2861
Numero di pagine14
RivistaJournal of Separation Science
Volume36
Stato di pubblicazionePubblicato - 2013

Keywords

  • PROTEINS

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