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

摘要: 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 internal sequence in agreement DNA-derived unknown coded D3Z9M3 (Swiss-Prot). To match the pattern and mass theoretical data, removal from N terminus signal peptide C three amino acid (a.a.) residues (Arg-Ala-Val) cyclization N-terminal glutamine pyroglutamic had to be supposed, resulting mature 90 a.a. trypsin digest ensured 100% coverage. For high content (34/90 37.8%) we propose name this gliadoralin A 1-90. Low amounts five different isoforms were sporadically detected, which did not significantly change their relative after stimulation. Gliadoralin is substrate for transglutaminase-2, having Lys 60 Gln as major determinants enzyme recognition. In silico investigation superior structures evidenced that small part adopts α-helical fold, whereas large segments are unfolded, suggesting unordered conformation.