Novel Cysteine Tags for the Sequencing of Non-Tryptic Disulfide Peptides of Anurans: ESI-MS Study of Fragmentation Efficiency |
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Authors: | Tatyana Y Samgina Egor A Vorontsov Vladimir A Gorshkov Konstantin A Artemenko Ilya E Nifant’ev Basem Kanawati Philippe Schmitt-Kopplin Roman A Zubarev Albert T Lebedev |
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Institution: | (1) Department of Chemistry, Moscow State University, Russian Federation, 119991 Leninskie Gory 1/3, Moscow, Russia;(2) Department of Physical and Analytical Chemistry, Uppsala University, Uppsala, Sweden;(3) GSF Institute of Ecological Chemistry, Neuherberg, Germany;(4) Department of Medicinal Biochemistry and Biophysics, Division of Molecular Biometry, Karolinska Institutet, Stockholm, Sweden; |
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Abstract: | Mass spectrometry faces considerable difficulties in de novo sequencing of long non-tryptic peptides with S–S bonds. Long
disulfide-containing peptides brevinins 1E and 2Ec from frog Rana ridibunda were reduced and alkylated with nine novel and three known derivatizing agents. Eight of the novel reagents are maleimide
derivatives. Modified samples were subjected to MS/MS studies on FT-ICR and Orbitrap mass spectrometers using CAD/HCD or ECD/ETD
techniques. Procedures, fragmentation patterns, and sequence coverage for two peptides modified with 12 tags are described.
ECD/ETD and CAD fragmentation revealed complementary sequence information. Higher-energy collisionally activated dissociation
(HCD) sufficiently enhanced y-ions formation for brevinin 1E, but not for brevinin 2Ec. Some novel tags N-benzylmaleimide, N-(2,6-dimethylphenyl)maleimide] along with known N-phenylmaleimide and iodoacetic acid showed high total sequence coverage taking into account combined ETD and HCD fragmentation.
Moreover, modification of long (34 residues) brevinin 2Ec with N-benzylmaleimide or N-(2,6-dimethylphenyl)maleimide yielded high sequence coverage and full C-terminal sequence determination with ECD alone. |
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