Method Development for the Detection of Human Myostatin by High-Resolution and Targeted Mass Spectrometry

Abstract

Myostatin, a highly conserved secretory protein, negatively regulates muscle development, affecting both the proliferation and differentiation of muscle cells. Proteolytic processing of the myostatin precursor protein generates a myostatin pro-peptide and mature protein. Dimerization of the mature myostatin protein creates the active form of myostatin. Myostatin dimer activity can be inhibited by noncovalent binding of two monomeric myostatin pro-peptides. This ability for myostatin to self-regulate as well as the altered expression of myostatin in states of abnormal health (e.g., muscle wasting) support the need for specific detection of myostatin forms. Current protein detection methods (e.g., Western blot) rely greatly on antibodies and are semiquantitative at best. Tandem mass spectometry (as in this study) provides a highly specific method of detection, enabling the characterization of myostatin protein forms through the analysis of discrete peptides fragments. Utilizing the scheduled high-resolution multiple reaction monitoring paradigm (sMRM; AB SCIEX 5600 TripleTOF) we identified the lower limit of quantitation (LLOQ) of both mature (DFGLDCDEHSTESR) and pro-peptide regions (ELIDQYDVQR) as 0.19 nmol/L. Furthermore, scheduled multiple reaction monitoring (sMRM; AB SCIEX QTRAP 5500) identified a LLOQ for a peptide of the pro-peptide region (LETAPNISK) as 0.16 nmol/L and a peptide of the mature region (EQIIYGK) as 0.25 nmol/L.

A targeted proteomic assay for the measurement of plasma proteoforms related to human aging phenotypes

“…The mean protein concentrations of both GDF8 mature protein and propeptide reported in the present study are somewhat similar to published GDF8 concentrations using ELISA [25][26][27][28][29] and immunoaffinity SRM [30][31][32]. Peiris and colleagues developed an SRM assay for GDF8 but did not report any values for human plasma [33].…”

A Sensitive and Selective Immunoassay for the Quantitation of Serum Latent Myostatin after In Vivo Administration of SRK-015, a Selective Inhibitor of Myostatin Activation

“…15 Prior to development of this assay, methods for directly measuring specific myostatin proforms in biological samples were limited to mass spectrometrybased technologies. [29][30][31][32] While mass spectrometry has become more accessible, ELISAs and plate-based immmunoassays offer higher throughput and lower cost, and they pose fewer technical challenges. Furthermore, unlike established myostatin ELISAs that require conversion of all myostatin forms to the mature form prior to analysis, [21][22][23][24][25][26] the assay described here directly measures latent myostatin, thereby reducing the sample manipulation required.…”

“…For these reasons, specific, quantitative pro-or latent myostatin measurement has thus far been achieved only by mass spectrometrybased methods. [29][30][31][32] Using antibodies directed at the myostatin prodomain, we previously developed a Western blot-based method to quantify relative changes in latent myostatin expression. 15 Because Western blots are semiquantitative and lowthroughput, however, we have subsequently developed a sensitive, robust, and highly selective plate-based ligand binding assay to directly measure latent myostatin.…”

Efeito da suplementação de leucina na sinalização da via da miostatina durante atrofia muscular esquelética.

“…A dimerização desse fragmento maduro de 12 kDa (através de pontes dissulfeto), é que cria a forma ativa da proteína, que tem aproximadamente 28 kDa. (ARGILES et al, 2012;PEIRIS, ASHMAN, et al, 2014).…”

“…Biossíntese e processamento da miostatina. Adaptado de Peiris et al (PEIRIS, ASHMAN, et al, 2014) Uma vez ativada, a miostatina desencadeia uma cascata de sinalização muito similar a outros membros da superfamília TGFβ (Figura 5). Ela apresenta uma grande afinidade pelo receptor de membrana activina RIIB (ActRIIB), o qual recruta, fosforila e ativa o receptor do tipo I (ALK4 e ALK5).…”