Ppsq 1

To determine the molecular mass and amino acid sequence of the purified SMP, it was analysed using an automated N‐terminal amino acid gas‐phase sequencer (PPSQ‐1; Shimadzu Corp. Nakagyo‐ku, Kyoto, Japan) and a MALDI‐TOF mass spectrometer (Voyager‐DE^™ PRO spectrometer; Perseptive Biosystem, Framingham, MA, USA). On the basis of the structural determination results, two peptides, with or without the carboxyl‐terminus amidated, were automatically synthesized by a conventional solid‐phase method with Fmoc‐protected amino acids and coupling reagents, 1‐hydroxybenzotriazole and N,N‐diisopropylcarbodimide, using a peptide synthesizer (PSSM‐8; Shimadzu) as described previously (Kim et al. 2015). Other neuropeptides, S1 (GFNSALMFamide), S2 (SGPYSFNSGLTFamide), FMRFamide and FLRFamide were synthesized to enable comparison of their activities with that of the identified peptide.

Proteolytic fragments were transferred onto 0.2 μm PVDF membranes (Applied Biosystems), using a Trans-Blot Turbo Transfer System (Biorad) in 10 min (25 V 1.3 A) and the transfer buffer: 50 mM Tris, 50 mM Boric Acid, 0.1% SDS, 10% Ethanol, pH 8.3. Bands corresponding to proteolytic polypeptides were excised after Coomassie blue staining.
N-terminal sequences for each proteolytic peptide were obtained by automated Edman degradation using a sequencer (PPSQ-1 Shimadzu). The PVDF membranes were washed three times in Ethanol/water (90/10), dried, and loaded in the cartridge of the Edman sequencer Shimadzu PPSQ 31B. Ten Edman degradation cycles were performed.