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<p>Platelets are pivotal circulating effector cells central to diverse physiological and pathological processes, with many of their essential functions—such as activation, adhesion, and signal transduction—being modulated by protein glycosylation. However, the inherent microheterogeneity and low ionization efficiency of glycopeptides continue to pose substantial technical barriers to achieving large-scale and precise analysis at the intact glycopeptide level, thereby limiting the in-depth exploration of platelet glycoproteome. In this study, we first evaluated three enzymatic digestion strategies—trypsin alone, Lys-C/trypsin combination, and Glu-C/trypsin combination, followed by optimization of mass spectrometry parameters for stepped-collision-energy high-energy collisional dissociation (sceHCD), ultimately establishing an efficient glycoproteomic workflow tailored to human peripheral blood platelets. Application of this workflow to platelets from healthy volunteers led to the successful identification and quantitation of 4696 intact N-glycopeptides mapping to 299 glycoproteins and 534 glycosites involving 447 distinct glycans. Extension to the O-glycoproteome further confirmed workflow generalizability, successfully identifying 2807 O-glycopeptides, 548 O-glycosites, and 340 O-glycoproteins. Exploratory functional analysis indicated that males were enriched with glycoproteins associated with coagulation and complement regulation, whereas for females pathways related to cell adhesion and viral infection response were enriched. These sex‑dimorphic glycoprotein signatures provide insights into sex‑related differences in platelet physiology.</p> Graphical Abstract <p></p>

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Strategies for in-depth and simultaneous analysis of N- and O-glycoproteome of platelets uncovering sex-specific signatures of human platelets

  • 期刊论文

  • 发表日期：

    2026年4月1日

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  • Guoqian Yang,
  • Jiaqi Yu,
  • Xinyu Zhao,
  • Aichao Wang,
  • Jian Zhang,
  • Zhuohan Du,
  • Yong Zhang,
  • Liujun Tang,
  • Linsheng Zhan,
  • Wantao Ying

摘要

Platelets are pivotal circulating effector cells central to diverse physiological and pathological processes, with many of their essential functions—such as activation, adhesion, and signal transduction—being modulated by protein glycosylation. However, the inherent microheterogeneity and low ionization efficiency of glycopeptides continue to pose substantial technical barriers to achieving large-scale and precise analysis at the intact glycopeptide level, thereby limiting the in-depth exploration of platelet glycoproteome. In this study, we first evaluated three enzymatic digestion strategies—trypsin alone, Lys-C/trypsin combination, and Glu-C/trypsin combination, followed by optimization of mass spectrometry parameters for stepped-collision-energy high-energy collisional dissociation (sceHCD), ultimately establishing an efficient glycoproteomic workflow tailored to human peripheral blood platelets. Application of this workflow to platelets from healthy volunteers led to the successful identification and quantitation of 4696 intact N-glycopeptides mapping to 299 glycoproteins and 534 glycosites involving 447 distinct glycans. Extension to the O-glycoproteome further confirmed workflow generalizability, successfully identifying 2807 O-glycopeptides, 548 O-glycosites, and 340 O-glycoproteins. Exploratory functional analysis indicated that males were enriched with glycoproteins associated with coagulation and complement regulation, whereas for females pathways related to cell adhesion and viral infection response were enriched. These sex‑dimorphic glycoprotein signatures provide insights into sex‑related differences in platelet physiology.

Graphical Abstract