title: 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 (sMRMHR; 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.
authors.affiliation: The University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital , Herston, Brisbane QLD 4029, Australia.
authors.forename: Hassendrini Nileishika
authors.initials: HN
authors.lastname: Peiris
authors.affiliation: The University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital , Herston, Brisbane QLD 4029, Australia.
authors.forename: Keith
authors.initials: K
authors.lastname: Ashman
authors.affiliation: The University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital , Herston, Brisbane QLD 4029, Australia.
authors.forename: Kanchan
authors.initials: K
authors.lastname: Vaswani
authors.affiliation: The University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital , Herston, Brisbane QLD 4029, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Kvaskoff
authors.affiliation: The University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital , Herston, Brisbane QLD 4029, Australia.
authors.forename: Gregory Edward
authors.initials: GE
authors.lastname: Rice
authors.affiliation: The University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital , Herston, Brisbane QLD 4029, Australia.
authors.forename: Murray David
authors.initials: MD
authors.lastname: Mitchell
keywords: GDF-8; Myostatin; mass spectrometry