Identifying N‐terminal peptides by a combination of the edman procedures with a bromine isotope tag: Application to the silicateins

• Published in: Protein science Vol. 25; no. 12; pp. 2277 - 2281
• Main Authors: Fedorova, G. A., Korneva, E. S., Belikova, A. S., Grachev, M. A.
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01-12-2016; John Wiley and Sons Inc
• Subjects: 4‐bromophenyl isothiocyanate (4‐Br‐PITC); Analogs; Animals; Band spectra; Bromine; Cathepsins; Cathepsins - chemistry; Cathepsins - genetics; Edman; Esters; Gene sequencing; Genetic transformation; Mass spectrometry; Mass spectroscopy; Methods and Applications; Peptides; Peptides - chemistry; Peptides - genetics; Porifera - genetics; Porifera - metabolism; Proteins; Residues; Sequence Analysis, Protein - methods; Silica; silicateins; Silicic acid; Silicon dioxide; Spicules; sponge; Sponges; Trifluoroacetic acid; Trypsinolysis; Edman; 4-bromophenyl isothiocyanate (4-Br-PITC); sponge; mass spectrometry; silicateins
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.3037

Silicateins are proteins found within spicules of siliceous sponges. They are analogs of proteinases cathepsins; they catalyze the transformation of silicic acid esters into biogenic silica (SiO2·nH2O), and are believed to take part in the processes of silicification in marine and freshwater sponges. Earlier studies by Kalyuzhnaya et al. revealed that the Baikal Sponge Lubomirskia baicalensis Pallas, 1773 (L. baicalensis) contains a gene 1988 bp long, which hosts four sequences that encode four mRNAs giving rise to silicateins α1, α2, α3 and α4 (SILα1, SILα2, SILα3, SILα4) whose predicted amino acid sequences are similar to those of the predicted sequences of marine sponge silicateins. However, the sequences of mature silicateins of L. baicalensis remained unknown, since their N‐terminal peptides were not identified. We found the sequences of these N‐terminal peptides using a combination of the Edman procedure, which involved reaction with phenylisothiocyanate, treatment with trifluoroacetic acid and trypsinolysis followed by treatment with 4‐bromine‐phenylisothiocyanate performed directly within polyacrylamide gel bands, and subsequent mass spectrometry. The N‐terminal peptides are YAESIDWR (SILα1), YVDSIDWR (SILα2 and α4), and YADSLDWR (SILα3). All mature silicateins of L. baicalensis had a length 217 amino acid residues.