Protein glycosylation is the most common epiproteomic modification involved in numerous physiological and pathological processes. Previous studies reported strong associations between human plasma N-glycans and age, prompting us to evaluate the potential application of this biological phenomenon in the field of forensics. Blood from 526 blood donors from different parts of Croatia was collected on bloodstain cards during the period 2004-2007 and stored at 4°C for 6-9 years. Glycosylation profiles of the bloodstains were analysed using hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC) and divided into 38 glycan groups (GP1-GP38). A statistically significant correlation between N-glycan profiles of bloodstains and chronological age was found and a statistical model that can be used for the age prediction was designed (Age = 75.59 - 5.15 × (GP4)(2)+ 17.07 × GP6 - 5.30 × (GP10)(2) - 16.56 × GP16 + 20.07 × GP20 - 7.54 × (GP20)(2) + 16.47 × GP22). This model explains 47.78% of the variation in age, with a prediction error of 9.07 years. Our findings demonstrate that analysing the N-glycan profile could be a new tool in forensics, offering an approximate human age estimation from dried bloodstains found at a crime scene.
