title: Novel cysteine tags for the sequencing of non-tryptic disulfide peptides of anurans: ESI-MS study of fragmentation efficiency.
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.
authors: Department of Chemistry, Moscow State University, Russian Federation, 119991 Leninskie Gory 1/3, Moscow, Russia.
authors: Tatyana Y
authors: TY
authors: Samgina
authors: Egor A
authors: EA
authors: Vorontsov
authors: Vladimir A
authors: VA
authors: Gorshkov
authors: Konstantin A
authors: KA
authors: Artemenko
authors: Ilya E
authors: IE
authors: Nifant'ev
authors: Basem
authors: B
authors: Kanawati
authors: Philippe
authors: P
authors: Schmitt-Kopplin
authors: Roman A
authors: RA
authors: Zubarev
authors: Albert T
authors: AT
authors: Lebedev
mesh_terms: D000595:Amino Acid Sequence; D029845:Amphibian Proteins / Q000737:chemistry*; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D005260:Female; D019807:Iodoacetic Acid / Q000737:chemistry; D008297:Male; D008301:Maleimides / Q000737:chemistry; D015335:Molecular Probes; D008969:Molecular Sequence Data; D011895:Rana ridibunda; D020539:Sequence Analysis, Protein; D012867:Skin / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D029845:Amphibian Proteins; D023181:Antimicrobial Cationic Peptides; D004220:Disulfides; D008301:Maleimides; D015335:Molecular Probes; D003545:Cysteine; D019807:Iodoacetic Acid