π-HelixNovo2: Making Accurate Online De Novo Peptide Sequencing Available to All

peer-reviewed · Genomics, Proteomics & Bioinformatics · 2026

peer-reviewed · Genomics, Proteomics & Bioinformatics · 2026. Tingpeng Yang et al. De novo peptide sequencing, the mainstream technique for identifying novel peptides, has recently seen…
Date 2026-06-25
Type peer-reviewed
Venue Genomics, Proteomics & Bioinformatics
Publisher Oxford University Press (OUP)
Contribution algorithm
DOI 10.1093/gpbjnl/qzag049
Citations (OpenAlex) 0
Venue 2-year citedness 5.77

Abstract

De novo peptide sequencing, the mainstream technique for identifying novel peptides, has recently seen remarkable improvements due to deep learning approaches. However, existing models struggle to effectively enhance the encoding of mass spectra and the decoding of amino acids, which limits their overall performance. Moreover, these models lack peptide filtering for de novo peptides, and often present challenges for users without programming expertise. Here, we propose π-HelixNovo2, a de novo peptide sequencing model that integrates complementary spectrum and bidirectional decoding within a Transformer framework. We further propose a peptide filtering strategy to identify the correct peptide-spectrum matches from the results of π-HelixNovo2. Our experiments demonstrate that π-HelixNovo2 outperforms state-of-the-art models, offering reliable performance in identifying antibody peptides, multi-enzyme cleavage peptides, non-enzymatic peptides, and analyzing the gut metaproteome. Finally, we trained π-HelixNovo2 on the large-scale MassIVE-KB dataset, and present an open, user-friendly, and online computational platform to make π-HelixNovo2 freely available to all (https://openi.pcl.ac.cn/OpenI/pi-HelixNovo-NPU).

Authors

  1. Tingpeng Yang · Peng Cheng Laboratory, Tsinghua Shenzhen International Graduate School, Tsinghua University
  2. Tianze Ling · Beijing Institute of Lifeomics, State Key Laboratory of Medical Proteomics, Tsinghua University
  3. Boyan Sun · Beijing Institute of Lifeomics, State Key Laboratory of Medical Proteomics
  4. Zhendong Liang · Peng Cheng Laboratory, Tsinghua Shenzhen International Graduate School, Tsinghua University
  5. Cheng Lai · Peng Cheng Laboratory
  6. Jiangli Hu · Peng Cheng Laboratory
  7. Zexuan Yi · Peng Cheng Laboratory
  8. Yonghong He · Peng Cheng Laboratory, Tsinghua University
  9. Leyuan Li · Beijing Institute of Lifeomics, State Key Laboratory of Medical Proteomics
  10. Yue Yu · Peng Cheng Laboratory
  11. Cheng Chang · Beijing Institute of Lifeomics, International Academy of Phronesis Medicine (Guangdong), National Center for Protein Sciences (Beijing), State Key Laboratory of Medical Proteomics
  12. Yu Wang · Peng Cheng Laboratory

Methods and tools

  • π-HelixNovo2: Successor to π-HelixNovo with an emphasis on availability: an online inference service alongside the model architecture refinement. Same Tsinghua / Pengcheng Lab / NCPSB collaboration as the original.

Data used

  • Anti-FLAG-M2 antibody sequencing (as deposited) · PXD023419
  • BALF proteomics - In-depth proteomic analysis of human bronchoalveolar lavage fluid towards the biomarker discovery for (as deposited) · PXD012645
  • BoxCar acquisition method enables single shot proteomics at a depth of 10,000 proteins in 100 minutes (as deposited) · PXD006109
  • Casanovo non-enzymatic fine-tuning set (as deposited) · MSV000094014
  • Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells (as deposited) · PXD012979
  • HeLa proteome of 12,250 protein-coding genes (as deposited) · PXD004452
  • High-resolution spatially-resolved proteome mapping using automated, sacrificial liquid-mediated sample transfer from la (as deposited) · PXD008844
  • Low-density lipoprotein receptor-related protein 1 (LRP1)-derived peptides protect against aggregation of LDL and choles (as deposited) · PXD011246
  • MassIVE-KB (v1) · MSV000081142
  • Mining waste streams of food production for bioactive plant polysaccharides that affect the fitness and expressed activi (as deposited) · MSV000082287
  • Monoclonal antibody de novo assembly (as deposited) · MSV000079801
  • NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin (as deposited) · PXD013233
  • Predictive signatures of 19 antibiotics-induced Escherichia coli proteomes (as deposited) · PXD016001
  • Proteome of the rodent malaria parasite Plasmodium berghei liver stage merosomes (as deposited) · PXD010559
  • Proteomic analysis of six different tissues from the Atlantic bottlenose dolphin (Tursiops truncatus) (as deposited) · PXD008808
  • The Proteome Landscape of the Kingdoms of Life (as deposited) · PXD014877, PXD019483
  • The beta subunit of nascent polypeptide associated complex plays a role in flowers and siliques development of Arabidops (as deposited) · PXD016315

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