Dystrophin Protein Quantification as a Duchenne Muscular Dystrophy Diagnostic Biomarker in Dried Blood Spots Using Multiple Reaction Monitoring Tandem Mass Spectrometry: A Preliminary Study

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle loss, leading to difficulties in movement. Mutations in the DMD gene that code for the protein dystrophin are responsible for the development of DMD disorder, where the synthesis of this protein is completely halted. Therefore, circulating dystrophin protein could be a promising biomarker of DMD disease. Current methods for diagnosing DMD have sensitivity, specificity, and reproducibility limitations. Herein, a quantitative liquid chromatography–tandem spectrometry (LC–MS/MS) technique in multiple reaction monitoring (MRM) mode was designed and validated for accurate dystrophin protein measurement in a dried blood spot (DBS). The method was successfully validated on the basis of international guidelines regarding calibration curves, precision, and accuracy. In addition, patients and healthy controls were used to test the amount of dystrophin protein circulating in DBS samples as a potential biomarker for DMD disorders. DMD patients were found to have considerably lower levels than controls. To the best of our knowledge, this is the first study to report dystrophin levels in DBS through LC–MS/MS as a diagnostic marker for DMD to the proposed MRM method, providing a highly specific and sensitive approach to dystrophin quantification in a DBS that can be applied in DMD screening.

A Proof of Principle Proteomic Study Detects Dystrophin in Human Plasma: Implications in DMD Diagnosis and Clinical Monitoring

“…This technique is considered one of the main analytical methods in proteome research, and is suitable for protein/peptide identification in body fluids [ 31 , 32 ]. Due to technological advances, it is becoming increasingly used for biomarker quantification in clinical assays [ 33 , 34 ] and was recently used to detect DYS in dried blood spots [ 23 ]. Moreover, recently, a targeted proteomic test for SARS-CoV-2 has become the first targeted proteomic clinical test UKAS accredited and MHRA approved for clinical use in patients [ 34 ].…”

“…[ 21 ] and is not considered a constitutively expressed protein detectable in body fluids [ 22 ]. Recently, two dystrophin peptides of unknown origin, were found in dry blood spots of DMD patients and healthy controls, by targeted liquid chromatography mass spectrometry [ 23 ]. Possibly, the DMD fragments identified in blood spots may come from blood cells, i.e., granulocytes and B-cells, since these cells are known to express dystrophin, based on RNA-sequencing results [ 24 ].…”