Urinary Proteome Profiling by Several Methods Identifies Titin as the Most-Differentiating Noninvasive Urinary Biomarker of Disease Severity in Becker Muscular Dystrophy

Figure 1. Peptide-spectrum matches (PSMs) of titin in BMD urine samples: box plot showing levels of urinary titin measured using label-free proteomic method. Healthy controls (n = 5); ambulatory BMD (n = 5), non-ambulatory BMD (n = 4) ** indicates significant adjusted p-value of 0.0013; reported using Tukey’s HSD post hoc analysis.

Figure 2. Levels of titin tryptic peptide measured by targeted mass spectrometry in urine samples of BMD patients and healthy controls. Titin tryptic peptide VPAPVEIPVTPPTLVSGLK was targeted for quantification using PRM and SILAC spike-in strategy. Data are plotted as log2 ratio of endogenous peptide to SILAC-labeled peptide. Healthy controls (n = 3); ambulatory BMD (n = 21), non-ambulatory BMD (n = 6). ** indicates significant p-value of 0.0018; reported using Tukey’s post hoc analysis.

Figure 3. Western blot detection of N-terminal titin (~30–32.8 kDa) in urine samples of healthy controls (n = 3), ambulatory BMD (n = 3) and non-ambulatory BMD (n = 3). Urine aliquot containing 15 µg of total proteins was processed for Western blot analysis as described in the Methods.

Figure 4. Box plots representing the levels of the N-terminal titin fragment in urine samples of ambulatory BMD (n = 20), non-ambulatory BMD (n = 10) and healthy controls (n = 10). (a) Non-normalized data; (b) data normalized to total protein concentration in urine samples. * p-value = 0.02; ** p-value = 0.0023; *** p-value = 0.0001; reported using Tukey’s HSD post hoc analysis.

Figure 5. (a) Titin/total protein adjusted for current age, ambulatory BMD (n = 21), non-ambulatory BMD (n = 6); (b) Titin/total protein adjusted for duration of disease, ambulatory BMD (n = 21), non-ambulatory BMD (n = 6); (c) Titin/total protein comparison between the disease severities based on age of onset, ambulatory BMD (0–5) (n = 9), non-ambulatory BMD (0–5) (n = 4); ambulatory BMD (6–10) (n = 5), non-ambulatory BMD (6–10) (n = 4); ambulatory BMD (11–16) (n = 2), non-ambulatory BMD (11–16) (n = 2); ambulatory BMD (>21) (n = 4). * indicates significant adjusted p-value of 0.0425; reported using Holm–Sidak post hoc analysis.