Peiris Hassendrini Nileishika, Ashman Keith, Vaswani Kanchan, Kvaskoff David, Rice Gregory Edward, Mitchell Murray David
The University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital , Herston, Brisbane QLD 4029, Australia.
J Proteome Res. 2014 Aug 1;13(8):3802-3809. doi: 10.1021/pr5004642. Epub 2014 Jun 30.
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.
肌生成抑制素是一种高度保守的分泌蛋白，对肌肉发育起负调节作用，影响肌肉细胞的增殖和分化。肌生成抑制素前体蛋白的蛋白水解加工产生肌生成抑制素前肽和成熟蛋白。成熟肌生成抑制素蛋白的二聚化产生肌生成抑制素的活性形式。两个单体肌生成抑制素前肽的非共价结合可抑制肌生成抑制素二聚体活性。肌生成抑制素的这种自我调节能力以及在健康异常状态（如肌肉萎缩）下肌生成抑制素表达的改变，支持了对肌生成抑制素不同形式进行特异性检测的必要性。目前的蛋白质检测方法（如蛋白质印迹法）在很大程度上依赖抗体，充其量只是半定量的。串联质谱法（如本研究中所用）提供了一种高度特异性的检测方法，能够通过分析离散的肽片段来鉴定肌生成抑制素蛋白的形式。利用预定的高分辨率多反应监测模式（sMRM；AB SCIEX 5600 TripleTOF），我们确定成熟区域（DFGLDCDEHSTESR）和前肽区域（ELIDQYDVQR）的定量下限（LLOQ）均为0.19 nmol/L。此外，预定多反应监测（sMRM；AB SCIEX QTRAP 5500）确定前肽区域的一个肽段（LETAPNISK）的LLOQ为0.16 nmol/L，成熟区域的一个肽段（EQIIYGK）的LLOQ为0.25 nmol/L。
