Background: Molecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools. Methods: Two technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS). Results Five, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml. Conclusions: These results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.

The serum samples were prepared semi-automatically. Firstly, 2 µl of serum was transferred into 50 mM AmBic on the CyBio® Selma liquid handler (Analytik Jena Ag, Germany) and thereafter 8 µl of QPrEST mastermix that represented a 1:1 (Light:Heavy, L:H) peptide ratio to the endogenous levels in plasma if possible was also added on the Selma. Then the proteins were denatured with 1% sodium deoxycholate (SDC) and reduced with 10 mM DTT for 20 min at 56 °C. Thereafter the samples were alkylated with 50 mM CAA and incubated in dark for 20 min. The proteins were digested with LysC and trypsin as described above with incubation times of 1 h and overnight, respectively. The digestion was quenched by adding TFA to a final concentration of 0.5% (v/v). SDC was precipitated for 30 min at room temperature and then centrifuged for 10 min at 3,273 rcf on Allegra X 12R centrifuge (Beckman-Coulter, Brea, CA, USA). The sample was cleaned as described previously (Edfors, et al 2018 ) by using in-house prepared StageTips packed with Empore C18 Binded Silica matrix (3M, Saint Paul, MN, USA). Desalted peptides were vacuum‐dried and stored at -20 °C before subjected for LC‐MS/MS analysis. An experiment with 7 replicates following the above-described sample preparation method was performed to ensure method suitability and repeatability across the sample plate. Liquid chromatography Samples were measured using the same LC-MS system as described above. The serum samples were stored lyophilised and resuspended by the autosampler. A total of 1 µg peptides were first loaded onto the trap column, washed 5 min at 5 μl/min with 100% of solvent A, and then separated by a PepMap RSLC C18 column (75 µm x 25 cm, 2 µm, 100 Å, Thermo Scientific). The peptides were eluted with a linear 33 min gradient of 3 30% solvent B at a flow rate of 0.400 µl/min followed by a 2 min increase to 43% and then 1 min increase to 99% of solvent B. Column was washed for 8 min with 99% of solvent B at a flow rate of 0.650 µl/min and then equilibrated for approximately 10 minutes min with 1% of solvent B at a flow rate of 0.400 µl/min. Parallel reaction monitoring For sample analysis previously developed PRM method was used. Each full MS scan at 60,000 resolution (AGC target 3e6, mass range 350-1,600 m/z and injection time 110 ms) was followed by 20 MS/MS scans at 60,000 resolution (AGC target 2e5, NCE 27, isolation window 1.5 m/z and injection time 300 ms). The PRM method was defined by a scheduled (3 min windows) PRM isolation list that contained 34 paired light and heavy peptide precursors.

83 serum samples from a longitudinal cohort consisting of 20 DMD patients and 18 healthy control patients, along with patient data, were collected at Leiden University Medical Center with ethical approval LUMC Commissie Medische Ethiek and the study approved by Regionala etikprövningsnämnden Stockholm, Sweden (ref. 2018/1859-31/1). Samples were aliquoted and divided between two laboratories for biomarker quantification. Total protein concentration of each sample was measured at 5 different dilutions using Pierce™ BCA Protein assay (23225, Thermo Scientific) according to the manufacturer’s instructions.