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Characterization of Performance-Enhancing Peptides via Inlet Ionization on DART-TOF/MS

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Description of original award (Fiscal Year 2013, $80,765)

While recombinant human growth hormone (rHGH) is prescribed legitimately to individuals suffering from growth hormone deficiency, it has obtained a more nefarious presence in cycling, bodybuilding and other physical competitions. Similarly to anabolic-androgenic steroids (AAS), rHGH is abused to artificially enhance performance. The increased presence and subsequent abuse of rHGH and related peptides has been observed as internet vendors illicitly selling these substances have created a market for not only professional competitors, but the general public. In order for successful prosecution of those responsible for possession, manufacture and distribution, it is essential that the performance enhancing peptides can be characterized in a common forensic setting, with a reasonable cost.At present, the published methods are largely insufficient for peptides other than rHGH. In addition, these methods require instrumentations that are not within the general scope of a drug chemistry laboratory.
Proposed herein is the development of a method suitable for a modern forensic drug chemistry laboratory. This method provides for three separate examinations: a simple ultraviolet-visible spectrophotometric (UV-Vis) examination to distinguish between suspected peptide-based substances and other chemical bulk powders which have divergent methods of analysis. This is followed by intact protein/peptide measurement using Direct Analysis in Real Time Time of Flight (DART-TOF) via inlet ionization. The protein/peptide yields multiple-charged peaks, which can be deconvoluted to calculate the exact mass and match with the theoretical value.Then, a traditional enzymatic digestion protocol will be utilized to digest the protein/peptide, and several parameters will be investigated targeting complete and reproducible result. Finally, the digested peptide fragments will again be measured by inlet ionization on DART-TOF. The exact masses of the peptide fragments will be deduced through deconvolution, and compared to the exact mass values of fragments with a theoretical digestion. A database will be compiled for both the intact and digested protein/peptide, serving as references for other interested laboratories.
Inlet ionization is an emerging ionization technology promising sensitive and precise measurement of large biological molecules. Researchers reported successful protein/peptide measurement using this technique based on modification of a variety of instruments. This is the first attempt to apply this technology on DART-TOF, and the ionization source only needs to be slightly modified to allow protein/peptide measurement. In our lab the intact protein of ubiquitin was measured within 3ppm mass difference using inlet ionization on DART-TOF.
To that end, this project aims to develop an efficient, standardized method of protein and peptide analysis suitable for a forensic drug chemistry laboratory. With the increasing utilization of DART-TOF in the forensic science community, such a method will greatly advance the reach of the criminal justice system in these types of drug possession, distribution, or manufacturing cases, as well as ensuring public safety. Method information and spectra will be compiled in a resource guide and distributed to the forensic science community electronically and in person at national forensic science/analytical chemistry meetings.

Date Created: September 8, 2013