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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
Author(s) -
Cabras Tiziana,
Iavarone Federica,
Pirolli Davide,
Cristina De Rosa Maria,
Vitali Alberto,
Faa Gavino,
Cordaro Massimo,
Messana Irene,
Ekström Jörgen,
Castagnola Massimo
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201300312
Subject(s) - chemistry , glutamine , biochemistry , peptide sequence , trypsin , peptide , microbiology and biotechnology , amino acid , enzyme , biology , gene
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 D 3 Z 9 M 3 ( S wiss‐ P rot). 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 ( A rg‐ A la‐ V al) 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 L ys 60 and different G ln 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.