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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.