Refining Platelet Purification Methods: Enhancing Proteomics for Clinical Applications

TL;DR

This study improves platelet isolation and proteomic analysis methods, enabling high-purity sample preparation and identifying LFQ-DIA as the most effective approach for comprehensive platelet proteomics, with implications for clinical biomarker research.

Contribution

The paper introduces an optimized platelet isolation protocol and compares proteomic methods, establishing LFQ-DIA as superior for platelet protein analysis.

Findings

Optimized isolation reduces contamination and platelet loss.

LFQ-DIA outperforms other proteomic approaches in coverage and sensitivity.

Age affects platelet proteome, emphasizing demographic matching.

Abstract

Background: Platelet proteomics offers valuable insights for clinical research, yet isolating high-purity platelets remains a challenge. Current methods often lead to contamination or platelet loss, compromising data quality and reproducibility. Objectives: This study aimed to optimize a platelet isolation technique that yields high-purity samples with minimal loss and to identify the most effective mass spectrometry-based proteomic method for analyzing platelet proteins with optimal coverage and sensitivity. Methods: We refined an isolation protocol by adjusting centrifugation time to reduce blood volume requirements while preserving platelet yield and purity. Using this optimized method, we evaluated three proteomic approaches: Label-free Quantification with Data-Independent Acquisition (LFQ-DIA), Label-free Quantification with Data-Dependent Acquisition (LFQ-DDA), and Tandem Mass Tag labeling with DDA (TMT-DDA). Results: LFQ-DIA demonstrated superior protein coverage and sensitivity compared to LFQ-DDA and TMT-DDA. The refined isolation protocol effectively minimized contamination and platelet loss. Additionally, age-related differences in platelet protein composition were observed, highlighting the importance of using age-matched controls in biomarker discovery studies. Conclusions: The optimized platelet isolation protocol provides a cost-effective and reliable method for preparing high-purity samples for proteomics. LFQ-DIA is the most suitable approach for comprehensive platelet protein analysis. Age-related variation in platelet proteomes underscores the need for demographic matching in clinical proteomic research.