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.
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