( e Procise cLC sequencing system ...)Efficient Primary Structure Elucidation of Disulfide-bridged Peptides

Highly accurate electrospray tandem mass spectrometry for the elucidation of disulfide-bridge patterns using the QSTAR Pulsar hybrid quadrupole time-of-flight mass spectrometer

Combination of two highly senisitive technologies provides information on complex disulfide structures

Methods

Sample Preparation: The multicomponent venom of the spider Cupiennius salei (Figure 1) was purified by a combination of gel filtration, cation exchange chromatography and reversephase HPLC1.

Edman sequencing: Two important neurotoxic peptides1,2,3, CSTX-1 (8'351.90 Da) and CSTX-9 (7'530.25 Da) were subjected to sequence determination by Edman degradation in a Procise cLC 492 protein sequencer. The reduced Efficient Primary Structure Elucidation of Disulfide-bridged Peptides Application Note Peptide Sequencing Tandem Mass Spectrometry www.appliedbiosystems.com Figure 1. Venom from Cupiennius salei was used in this study. and alkylated samples were sequenced up to Ile 53 (CSTX-1) and Ala 47 (CSTX-9), respectively. In order to secure the rest of the sequences, reduced and alkylated CSTX-1 was cleaved with endoproteinase Asp-N and chymotrypsin, CSTX-9 with endoproteinase Asp-N and immobilised trypsin. In each case the peptides generated were separated by RP-HPLC and identified by amino acid analysis and mass spectrometry. Native CSTX-1 and CSTX-9 were cleaved with immobilised trypsin to eliminate disulfide exchange during digestion, and iodoacetamide was added as a scavenger to alkylate any free thiol groups that may have been formed during cleavage. The tryptic peptides were separated by RP-HPLC and the disulfide-containing peptides were identified by amino acid analysis and by tandem mass spectrometry. Electrospray tandem mass spectrometry: Identification of the disulfide-bridge pattern was performed by nano-electrospra
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