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Top‐down HPLC–ESI‐MS characterization of rat gliadoralin A, a new member of the family of rat submandibular gland glutamine‐rich proteins and potential substrate of transglutaminase
- Cabras, Tiziana, Iavarone, Federica, Pirolli, Davide, Cristina De Rosa, Maria, Vitali, Alberto, Faa, Gavino, Cordaro, Massimo, Messana, Irene, Ekström, Jörgen, Castagnola, Massimo
- Journal of separation science 2013 v.36 no.17 pp. 2848-2861
- glutamine, isoprenaline, protein-glutamine gamma-glutamyltransferase, proteins, rats, saliva, signal peptide, trypsin
- 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.