Label-free SERS Discrimination of Native Proline Hydroxylation at Single-molecule peptide by Deep Learning-assisted plasmonic nanopore

Yingqi Zhao; Kuo Zhan; Pei-Lin Xin; Yuge Liang; Enock Adjei Agyekum; Matti Putkonen; Shuai Li; Francesco De Angelis; Jianan Huang

arXiv · 2605.21084

Label-free SERS Discrimination of Native Proline Hydroxylation at Single-molecule peptide by Deep Learning-assisted plasmonic nanopore

Yingqi Zhao·Kuo Zhan·Pei-Lin Xin·Yuge Liang·Enock Adjei Agyekum·Matti Putkonen·Shuai Li·Francesco De Angelis·Jianan Huang

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Abstract

Post-translational modifications (PTMs) play essential roles in regulating protein structure, function, and cellular signalling. However, peptide level discrimination of hydroxylation at the single-molecule level remains difficult. Here, we report a particle-in-pore single-molecule surface-enhanced Raman spectroscopy (SERS) platform combined with peak occurrence frequency (POF) analysis and a one-dimensional convolutional neural network (1D-CNN) for discriminating hydroxylated and non-hydroxylated HIF peptide fragments. Three peptide pairs containing the Pro-564 hydroxylation site, with lengths of 7, 9, and 15 amino acids (AAs), were investigated. POF analysis revealed reproducible hydroxylation-dependent spectral changes in the 7AA and 9AA peptide pairs, which were attributed to changes in adsorption conformation and surface interactions. CNN-based classification achieved post-evaluation accuracies of 72.98%, 78.55%, and 89.74% for the 7AA, 9AA, and 15AA peptide pairs, respectively, with AUC values above 0.80 for all the pairs, indicating a reliable discrimination. Gradient-weighted feature visualization further showed that CNN-sensitive regions overlapped with recurrent POF features, supporting the chemical relevance of the learned classification patterns. Notably, for the 15AA peptide pair, the enhanced citrate-associated band suggests that hydroxylation can substantially alter peptide-gold nanoparticle adsorption behaviour. This adsorption-mediated effect may amplify hydroxylation-induced spectral differences and contribute to the improved discrimination accuracy despite the increased structural complexity. These results demonstrate that the particle-in-pore sensor, assisted by deep learning, can capture hydroxylation-induced spectral and adsorption changes in peptide fragments, providing a promising strategy for ultrasensitive analysis of weak PTM signatures in peptides.

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Yingqi Zhao, Kuo Zhan, Pei-Lin Xin, Yuge Liang, Enock Adjei Agyekum, Matti Putkonen, Shuai Li, Francesco De Angelis, Jianan Huang. 2026-05-20. Label-free SERS Discrimination of Native Proline Hydroxylation at Single-molecule peptide by Deep Learning-assisted plasmonic nanopore. https://arxiv.org/abs/2605.21084

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