Mass spectrometric detection of Myostatin Inhibitory Peptide 7, Kisspeptin-10 and analogues

Code: 22B07CR

For the detection of prohibited peptides hormones according to WADA Probibited List 2022 [1] chapter S2, subchapters 2 (“Peptide hormones and their releasing factors”), 3 (“Growth factors and growth factor modulators”) and 4 (“Metabolic modulators”), mass spectrometric (MS) methods are used in combination with high performance liquid chromatography (HPLC). Typically, two different strategies are applied for sample preparation: small peptides (molecular mass below ca. 2 kDa, e.g. GH-releasing peptides (GHRPs), TB-500) are enriched from urine by solid phase extraction (SPE).[2] Larger peptides (mass above ca. 2 kDa, e.g. growth hormone-releasing hormone (GHRH) and its analogues, IGF-1 and its analogues, MGFs, insulins) require pre-concentration (e.g. ultrafiltration) followed by immunoaffinity extraction (e.g. immunoprecipitation with antibody-coated magnetic beads).[3]Frequently, the extracted peptides are analyzed in two separate screening procedures using LC-MS methods.[2-3]

Many of the peptides misused for doping purposes have not yet been pharmaceutically approved and are available only on the black market. Recently, two new substances were found there: Myostatin inhibitory peptide 7 (not to be confused with myostatin propeptide) – it contains 23 amino acids (WRQNTRYSRIEAIKIQILSKLRL-NH2; monoisotopic mass: 2954.741 Da) and effectively inhibits myostatin,[4-5] and Kisspeptin-10, which consists of only 10 amino acids (YNWNSFGLRF-NH2; monoisotopic mass: 1301.631 Da) and is an effective LH secretagogue, which evokes rapid and potent increases of LH and testosterone secretion.[6-7] To include the two

compounds and their analogues in the current screening procedures for small and large peptides, (1) a multiplexed immunoprecipitation protocol will be developed for Myostatin inhibitory peptide 7 and analogues in order to detect it together with the other large peptides, and (2) an SPE method for Kisspeptin-10 and analogues for detection with the other small peptides. Subsequently, extracts will be analyzed by nano-HPLC and high resolution high accuracy mass spectrometry. Analogues of both peptides will be custom-synthesized and made available to other doping control laboratories.