Mobile phases were (A) water with 0.1% formic acid and (B) acetonitrile also with 0.1% formic acid. antibodies, a reduction of the dystrophin transmission is detected in a small cohort of plasma samples from DMD patients when compared to healthy controls, female carriers, and other neuromuscular diseases. We also demonstrate the detection of dystrophin protein by an antibody-independent method using targeted liquid chromatography mass spectrometry. This last assay detects three different dystrophin peptides in all healthy individuals analysed and supports our finding that dystrophin protein is usually detectable in plasma. The results of our proof-of-concept study encourage further studies in larger sample cohorts to investigate the value of dystrophin protein as a low invasive blood biomarker for diagnostic screening and clinical monitoring of DMD. Keywords: DMD, dystrophin protein, plasma assay, immunoassay, LC-MS/MS 1. Introduction Dystrophinopathies are progressive, X-linked muscular disorders characterised by progressive muscle mass degeneration and weakness. They include three main clinical phenotypes: the most severe Duchenne muscular dystrophy (DMD) (OMIM # 310200), the milder Becker muscular dystrophy SIRT1 (BMD) (OMIM # 300376), and the isolated dilated cardiomyopathy (OMIM # 302045) [1]. Dystrophinopathies are caused by mutations in the dystrophin gene (= 5) were precipitated with ammonium sulphate to deplete the majority of albumin. Next, 30 L of plasma was aliquoted Pomalidomide (CC-4047) with 10 L of 30 g/L of the MassPREP enolase digestion standard protein (Waters Corp.) used here as a generic internal standard to control for the first sample preparation before using PrESTs. The samples were precipitated with 60 L of 3M ammonium sulphate before being sonicated for 10 min and centrifugated at high speed for 10 min. The supernatants were taken out, and pellets were washed again with 250 L of 1 1.8 M ammonium sulphate. After centrifugation, the pellets were resuspended into 40 L digest buffer composed of 100 mM Tris, Pomalidomide (CC-4047) pH 7.8, 6 M Urea, 2 M Thiourea, and 2% ASB14 and mixed for 10 min. Then, 3L of DTT answer (DL-Dithiothreitol DTT) at a concentration of 162 mM in 100 mM Tris-HCL, pH 7.8 was added. After incubation at room temperature for one hour, 6 Pomalidomide (CC-4047) L of 162 mM Iodoacetamide made in 100 mM Tris-HCL, pH 7.8 was added. Samples were shaken and incubated at room heat for one hour in the dark. A total of 330 L of ddH2O was added to dilute the urea before adding 10 L of sequencing grade altered trypsin (Promega, Madison, WI, USA) answer (1 mg/mL in 50 mM Acetic Acid). Samples were incubated for 16 h at 37 C. Finally, digests were washed and desalted by SPE using a 100 mg Bond Elut C18 96 well plate (Agilent, Santa Clara, CA, USA). Samples were resuspended in 0.2% trifluoroacetic acid (TFA). Wells were primed using 1 mL of 60% Acetonitrile and 0.1% TFA followed by two equilibration washes of 1 1 mL of 0.1% TFA. After loading the samples, Pomalidomide (CC-4047) the wells were washed using Pomalidomide (CC-4047) 1 mL of 0.1% TFA twice. Peptides were eluted using 0.5 mL of 60% Acetonitrile 0.1% TFA and dried using a centrifugal evaporator. Samples were resuspended in 50 L 3% Acetonitrile, 0.1% TFA before LC-MS/MS analysis. Samples were analysed using a XevoTM TQ-XS MS coupled to an ACQUITYTM UPLCTM I-Class chromatography system in electrospray positive ionization (Waters corporation). Digested plasma peptides were separated using an ACQUITYTM Premier Peptide BEH C18 Column, 300 ?, 1.7 m, 2.1 50 mm. Mobile phone phases were (A) water with 0.1% formic acid and (B) acetonitrile also with 0.1% formic acid. The runs were performed over a 15 min gradient. Starting conditions were 95% A and 5% B for 5 min. Then, between 5 and 12.90 min, A decreased to 80% and B increased to 20%. From 12.90 min to 13.20, A decreased to 70% and B increased to 30%. After 13.20.