Tandem Mass Tag-Based Serum Proteome Profiling for Biomarker Discovery in Young Duchenne Muscular Dystrophy Boys

Abstract

Blood-accessible molecular biomarkers are becoming highly attractive tools to assess disease progression and response to therapies in Duchenne muscular dystrophy (DMD) especially in very young patients for whom other outcome measures remain subjective and challenging. In this study, we have standardized a highly specific and reproducible multiplexing mass spectrometry method using the tandem mass tag (TMT) strategy in combination with depletion of abundant proteins from serum and high-pH reversed-phase peptide fractionation. Differential proteome profiling of 4 year-old DMD boys (n = 9) and age-matched healthy controls (n = 9) identified 38 elevated and 50 decreased serum proteins (adjusted P < 0.05, FDR <0.05) in the DMD group relative to the healthy control group. As expected, we confirmed previously reported biomarkers but also identified novel biomarkers. These included novel muscle injury-associated biomarkers such as telethonin, smoothelin-like protein 1, cofilin-1, and plectin, additional muscle-specific enzymes such as UTP-glucose-1-phosphate uridylyltransferase, aspartate aminotransferase, pyruvate kinase PKM, lactotransferrin, tissue alpha-l-fucosidase, pantetheinase, and ficolin-1, and some pro-inflammatory and cell adhesion-associated biomarkers such as leukosialin, macrophage receptor MARCO, vitronectin, galectin-3-binding protein, and ProSAAS. The workflow including serum depletion, sample processing, and mass spectrometry analysis was found to be reproducible and stable over time with CV < 20%. Furthermore, the method was found to be superior in terms of specificity compared to other multiplexing affinity-based methods. These findings demonstrate the specificity and reliability of TMT-based mass spectrometry methods in detection and identification of serum biomarkers in presymptomatic young DMD patients.

Figure 1

Chart depicting the overall workflow from sample preparation to mass spectrometry analysis to data processing. Serum samples (900 μg per aliquot) from 4 year-old DMD patients (n = 9) and age-matched healthy controls (n = 9) were processed for 12 most abundant proteins depletion. Resulting samples were in-solution-digested and randomized for TMT tagging, as shown in the figure. One of the control samples (dark blue) was used as a reference to normalize the data. Each sample mixture was further fractionated using a high-pH reversed-phase column and each fraction was analyzed by LC–MS/MS. Data were processed as described in the method.

Figure 2

Quality control of the immunoaffinity depletion of the 12 highly abundant proteins from serum samples. Aliquots of serum samples with different amounts of total proteins (300, 600, and 900 μg) were used in triplicate to examine the variability and quality of the immunoaffinity depletion process. (A) Linear response between the different starting amounts of total serum proteins and recovered total proteins in depleted samples. Approximately 17.9 μg (CV 1.1%), 49.9 μg (2.9%), and 81.4 μg (CV 7.3%) of proteins were recovered from serum samples containing 300, 600, and 900 μg of total proteins, respectively. (B) SDS-PAGE showing side-by-side nondepleted serum (3 μg) and depleted serum samples in triplicate (3 μg each). The nondepleted serum proteins exhibited very few intense protein bands, among which human serum albumin (HSA) was the most intense band on the 1D SDS-PAGE, while the depleted serum samples revealed large and reproducible number on new protein bands (ex. bands a, b, c, d, and e). (C) Detailed optical density analysis of randomly selected new protein bands observed on the depleted serum 1D SDS-PAGE (bands a, b, c, d, and e) allowed the comparison of the technical triplicate depletion of serum and estimated the CV < 9.1%. (D) Western blot analysis of depleted proteins (haptoglobin, HP) and nondepleted proteins (gelsolin, GSN). (E) Quantitative analysis of western blot shows an increase in GSN by approximately 10-fold (CVs < 16%) in comparison to nondepleted serum and a decrease in HP by approximately 2-fold (CVs < 14%), confirming the effectiveness and the reproducibility of the depletion kits. (F) Quality control of in-solution digestion and TMT tagging efficiency. A total of 618 serum proteins were identified and quantified. The coefficient of variation between these three technical replicates is depicted in the pie chart with the majority of quantified proteins (97%) showing CV < 20%, with 64% of proteins showing CV < 5% and 95% of proteins showing CV < 15%.

Figure 3

Data analysis results and panel of new protein biomarkers strongly associated to DMD. (A) Volcano plot showing significance vs log10 fold change in protein levels in serum of young DMD patients compared to healthy controls. Serum proteome profiling was performed on 4 year-old DMD patients (n = 9) and age-matched healthy controls (n = 9) using the TMT method. Significance was defined between the two groups by independent two-sample t-test (two-sided) corrected with adjusted P value and by permutation-based FDR of 0.05. Significantly elevated and significantly decreased proteins in DMD patients relative to controls are presented in green and red, respectively. (B) Hierarchical clustering of serum proteins whose levels were significantly altered between the 4 year-old DMD (n = 9) group and age-matched healthy control (n = 9) group. Intensities of proteins are normalized abundance ratios, which were log2-transformed. (C) Four new biomarkers in the cytoskeleton and sarcolemma protein category (PLEC, SMTNL1, TACP, and TNNC2) elevated in DMD group vs HC group. (D) Three new biomarkers in the enzyme protein category (PYGM, PFKM, and PKM) elevated in DMD group vs HC group. (E) Four new protein biomarkers involved in cytoskeleton structure and dynamics and extracellular matrix organization (COLT1, CFL1, SPON1, and TM8B4X) decreased in DMD group vs healthy control (HC) group. (F) Three new protein biomarkers in the enzyme or receptor protein category (CLN2, PIK3CB, and ERBB3) decreased in DMD group vs HC group. Box plots are showing the distribution of log2-transformed normalized protein abundance ratios in DMD group vs healthy control (HC) group.

Figure 4

Global correlation map of the top serum biomarkers associated with DMD status and ELISA analysis of a set of new biomarkers. (A) Pairwise correlation of 59 protein biomarkers measured across all 18 participants (9 DMD patients compared to 9 age-matched healthy controls) resulted in a matrix of correlation coefficients where each variable is compared to all others for DMD (protein name are in red) and healthy control protein names are in blue). Positive correlations are indicated in red rectangle, while negative correlations are indicated in blue rectangle. (B) The magnified area highlights biomarkers that are positively correlated in DMD patients. These same biomarkers did not show any correlation or showed negative correlations in healthy controls. (C) The magnified area highlights biomarkers that are negatively correlated in DMD patients, while these same biomarkers did not show any correlation in healthy subjects. (D) Comparative box plot of CFL1 in DMD patient vs healthy control study for ELISA and MS method. (E) Comparative box plot of FCN1 in DMD patient vs healthy control study for ELISA and MS method. (F) Comparative box plot of PLEC analysis in DMD patient vs healthy control study for ELISA and MS method.