Protein glycosylation modification as a novel forensic biomarker for discriminating monozygotic twins

Discriminating monozygotic (MZ) twins remains a significant challenge in forensic science due to their nearly identical DNA, which renders conventional short tandem repeat (STR) typing ineffective. Protein glycosylation, influenced by environmental factors, offers a promising alternative for personal identification. While epigenetic markers have shown potential in distinguishing MZ twins, many are unstable, prone to degradation, or unsuitable for forensic field samples. A stable and reliable biomarker is urgently needed to address this gap. This study uniquely explores protein glycosylation modifications as a novel approach to differentiate MZ twins. Each twin pair was randomly split into group A and group B. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blot (WB) analysis, we investigated N-glycosylation and O-glycosylation patterns in peripheral blood samples from six unrelated individuals and three pairs of MZ twins. LC-MS/MS analysis identified 720 distinct N-glycosylation-modified peptides across the three pairs of MZ twins, with 77 peptides unique to group A and 50 to group B. WB analysis successfully distinguished both six unrelated individuals and three pairs of MZ twins based on O-GlcNAc expression patterns, despite subtle differences within the twin pairs. Collectively, our findings demonstrate that protein glycosylation modifications can serve as reliable biomarkers for differentiating MZ twins, offering a significant advancement in forensic identification where traditional methods fall short. However, further validation in larger datasets and under simulated forensic conditions is necessary before widespread adoption. This study not only enriches the pool of potential forensic biomarkers but also opens new avenues for research into the role of glycosylation in individual specificity and its broader applications in personalized medicine and disease diagnostics.