Towards the standard-module approach to disulfide-linked polypeptide nanostructures. I. Methodological prerequisites and mass spectrometric characterization of the test two-loop structure.
Potentially biologically-active nanostructures can be created from single chains of unmodified peptides by cross-linking different regions of the chain by disulfide bonds and cleaving the chain at specified sites to obtain the final configuration. The availability of techniques for assembly and characterization of such structures was tested on a two-loop structure created from a 21-residue linear peptide. Directed intra-molecular disulfide bond formation was performed by inserting partial sequences favoring intra-molecular SS bond formation ("loops") separated by partial sequences disfavoring such a process ("spacers") into the precursor sequence. Peptide bond cleavage by partial acid hydrolysis at specific sites (GG, NP/DP) inside the loops opened them; the same process in the spacer separated the loops. Synthesis, oxidation and bond cleavage were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). The hydrolysis fragments of the produced nanostructures were characterized by tandem electrospray ionization Fourier transform mass spectrometry (ESI FT-MS) with collisional and electron capture dissociations. The latter technique was especially useful as it cleaves SS bonds preferentially. The feasibility of the proposed synthesis approach and the adequacy of the analysis techniques for the test structure were demonstrated.
Department of Chemistry, University of Southern Denmark, Odense, Denmark.
O A
OA
Mirgorodskaya
K F
KF
Haselmann
F
F
Kjeldsen
R A
RA
Zubarev
P
P
Roepstorff
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D006868:Hydrolysis; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary
D004220:Disulfides; D010446:Peptide Fragments; D010455:Peptides