Peptide characterization was achieved by means of analytical RP-HPLC (see above for the linear precursor), mass spectrometry, and amino acid analysis.
The peptide concentration as well as the amino acid composition were analyzed using an LC 3000 system from Eppendorf-Biotronik (Hamburg, Germany). Peptide hydrolysis was carried out in 6 N HCl at 110 °C in sealed tubes for 24 h. Subsequently, the hydrolyzed peptides were dried in a vacuum concentrator and redissolved. The respective amino acid concentrations were determined by comparison with an amino acid standard solution (Laborservice Onken, Gründau, Germany).
Peptide masses were determined using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. As matrix 2,5-dihydroxyacetophenone (2,5-DHAP) was used according to the manufacture’s guideline (Bruker Daltonics, Bremen, Germany). MALDI mass spectra were produced on an UltrafleXtreme instrument (Bruker Daltonics, Bremen, Germany).
The disulfide connectivity of the N-terminal fragment was determined as described recently [29 (link),30 (link)]. Therefore, a micrOTOF-Q III device (Bruker Daltonics, Bremen, Germany) was used to measure electrospray ionization (ESI) mass and tandem-mass spectra.
The peptide concentration as well as the amino acid composition were analyzed using an LC 3000 system from Eppendorf-Biotronik (Hamburg, Germany). Peptide hydrolysis was carried out in 6 N HCl at 110 °C in sealed tubes for 24 h. Subsequently, the hydrolyzed peptides were dried in a vacuum concentrator and redissolved. The respective amino acid concentrations were determined by comparison with an amino acid standard solution (Laborservice Onken, Gründau, Germany).
Peptide masses were determined using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. As matrix 2,5-dihydroxyacetophenone (2,5-DHAP) was used according to the manufacture’s guideline (Bruker Daltonics, Bremen, Germany). MALDI mass spectra were produced on an UltrafleXtreme instrument (Bruker Daltonics, Bremen, Germany).
The disulfide connectivity of the N-terminal fragment was determined as described recently [29 (link),30 (link)]. Therefore, a micrOTOF-Q III device (Bruker Daltonics, Bremen, Germany) was used to measure electrospray ionization (ESI) mass and tandem-mass spectra.