Detection of follistatin‐based inhibitors of the TGF‐β signaling pathways in serum/plasma by means of LC‐HRMS/MS and Western blotting

Abstract

Cytokines of the transforming growth factor beta (TGF‐β) superfamily such as myostatin and activin A are considered as key regulators of skeletal muscle mass. In vivo, their activity is controlled by different binding proteins such as follistatin (FST), whose interaction with the circulating growth factors prevents activation of the activin type II receptors. FST‐based protein therapeutics are therefore not only promising drug candidates for the treatment of muscular diseases but also potential performance‐enhancing agents in sports. Within this study, two complementary detection assays for FST‐based inhibitors of the TGF‐β signaling pathways in doping control serum and plasma samples were developed by using both monomeric FST and dimeric FST‐Fc fusion proteins as model compounds. The initial testing procedure is based on immunoaffinity purification, tryptic digestion, and LC‐HRMS/MS, offering high specificity by targeting tryptic signature peptides of FST. As the glycoprotein is also produced endogenously, the confirmation method employs immunoaffinity purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blotting in order to detect the intact proteins and differentiate synthetic FST‐Fc constructs from naturally occurring FST isoforms. Both assays were found to be highly specific with an estimated detection limit of 10 ng/ml. Moreover, a commercial sandwich enzyme‐linked immunosorbent assay was used to determine endogenous FST values. The detected FST serum levels of healthy volunteers were found below 5 ng/ml, which is in accordance with reference values from the literature and below the doping control detection methods' limit of detection (LOD). The presented assays expand the range of available tests for emerging doping agents, and the initial testing procedure can readily be modified to include further protein drugs.

Olympic Anti-Doping Laboratory: The Analytical Technological Road From 2016 Rio De Janeiro to 2021 Tokyo

“…However, Pfizer has announced that it is terminating two clinical studies evaluating domagrozumab for the treatment of DMD because it did not meet its primary efficacy end point [60]. The doping control laboratory in Cologne developed the detection of follistatin-based inhibitors of TGF-β metabolic reactions [61]. A thorough review has been published also by Cologne laboratory on this subject [62].…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…In the absence of authentic administration studies, detection windows are not available yet, but the presence of oligomeric follistatins in recombinant products suggests prolonged serum half‐lives, which results in serum being the preferred test matrix for anti‐doping purposes. Complementary to this approach, Walpurgis et al presented an assay utilizing bottom‐up LC‐MS/MS analyses for the detection of follistatin‐based inhibitors of the TGF‐β signaling pathway 145 . Here, serum samples were immunoaffinity purified, diagnostic peptides were generated by trypsin digestion, and the target peptides were detected using LC‐HRMS/MS.…”

“…In addition to myostatin‐binding proteins, also myostatin‐neutralizing antibodies are prohibited, and the capability of a test method to detect one representative (domagrozumab) and related compounds was recently demonstrated 146 . Applying a similar strategy as reported above by Walpurgis et al, 145 serum was immunopurified, and domagrozumab was trypsin digested to yield diagnostic peptides specific for this recombinant humanized monoclonal antibody. The peptides, originating from the antibody's light and heavy chain, were separated on a C‐8 analytical column (50 × 3 mm, 2.7 μm particle size) with 0.1% formic acid and acetonitrile (containing 0.1% formic acid and 1% DMSO) as solvents A and B, respectively.…”

“…[126][127][128] Conversely, way. 145 Here, serum samples were immunoaffinity purified, diagnostic peptides were generated by trypsin digestion, and the target peptides were detected using LC-HRMS/MS. Using a C-8 analytical column (50 × 3 mm, 2.7 μm particle size) and 0.1% formic acid as well as acetonitrile (containing 0.1% formic acid and 1% DMS) as solvents A and B, respectively, the peptides were chromatographed prior to ESI (positive mode) and product ion scan detection (resolution 30,000).…”

A Novel c.100C > G Mutation in the FST Gene and Its Relation With the Reproductive Traits of Awassi Ewes

“… 6 The primary function of FST is to bind and neutralize activin through its binding properties. 9 , 10 Since FST binds to activin, it inhibits follicle-stimulating hormone (FSH) secretion by blocking its receptor (Act-RII) from combining with it. 8 , 11 Through suppressing FSH secretion from the anterior pituitary, FST controls granulosa cell differentiation, follicular growth, atresia, and luteinization processes.…”