Sequence Elucidation of an Unknown Cyclic Peptide of High Doping Potential by ETD and CID Tandem Mass Spectrometry

Abstract

Identification of an unknown substance without any information remains a daunting challenge despite advances in chemistry and mass spectrometry. However, an unknown cyclic peptide in a sample with very limited volume seized at a Pennsylvania racetrack has been successfully identified. The unknown sample was determined by accurate mass measurements to contain a small unknown peptide as the major component. Collision-induced dissociation (CID) of the unknown peptide revealed the presence of Lys (not Gln, by accurate mass), Phe, and Arg residues, and absence of any y-type product ion. The latter, together with the tryptic digestion results of the unusual deamidation and absence of any tryptic cleavage, suggests a cyclic structure for the peptide. Electron-transfer dissociation (ETD) of the unknown peptide indicated the presence of Gln (not Lys, by the unusual deamidation), Phe, and Arg residues and their connectivity. After all the results were pieced together, a cyclic tetrapeptide, cyclo[Arg-Lys-N(C 6 H 9 )Gln-Phe], is proposed for the unknown peptide. Observations of different amino acid residues from CID and ETD experiments for the peptide were interpreted by a fragmentation pathway proposed, as was preferential CID loss of a Lys residue from the peptide. ETD was used for the first time in sequencing of a cyclic peptide; product ions resulting from ETD of the peptide identified were categorized into two types and named pseudo-b and pseudo-z ions that are important for sequencing of cyclic peptides. The ETD product ions were interpreted by fragmentation pathways proposed. Additionally, multi-stage CID mass spectrometry cannot provide complete sequence information for cyclic peptides containing adjacent Arg and Lys residues. The identified cyclic peptide has not been documented in the literature, its pharmacological effects are unknown, but it might be a "designer" drug with athletic performance-enhancing effects.

A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry

“…DALDA and dmtDALDA are 40 times more potent than morphine at inducing antinociception in mouse models, and may be misused as analgesic agents in sports. Additionally, dmtDALDA was identified in a confiscated vial from a racetrack in North America . Thus, it is highly desirable to develop a comprehensive analytical method capable of detecting very hydrophilic peptides as well as other peptides.…”

“…Additionally, dmtDALDA was identified in a confiscated vial from a racetrack in North America. 21,22 Thus, it is highly desirable to develop a comprehensive analytical method capable of detecting very hydrophilic peptides as well as other peptides.…”

Identification and Structural Characterization of Naturally-Occurring Broad-Spectrum Cyclic Antibiotics Isolated from Paenibacillus

“…The development of data analysis approaches to analyze complicated MS data is beneficial and will hopefully lead to the discovery and identification of novel compounds. Other approaches have included using specific MS techniques to aid in the identification of primary sequence assignments either by using a combination of different types of fragmentation data [24] or by sequential fragmentation to reduce the spectral complexity produced from multiple ring openings [25].…”

Synthesis and characterization of the N‐terminal acetylated 17‐23 fragment of thymosin beta 4 identified in TB‐500, a product suspected to possess doping potential

“…The use of performance‐enhancing peptides is a growing issue in human sports drug testing as well as in equine sports drug testing …”

“…[1] The use of performance-enhancing peptides is a growing issue in human sports drug testing [2][3][4][5] as well as in equine sports drug testing. [6] A product called TB-500, claimed to increase muscle growth and tissue repair in horses and other mammals, is available on the Internet and officially distributed. It is presented as 'the synthetic peptide of the active region of thymosin beta 4 (Tb4)', without any further qualitative description such as amino acid sequence or molecular weight.…”