The miniaturized isolation of neutrophil granules (MING) method allowed a deep proteome mapping of human neutrophil granules

Author(s)

Alexandria, Gabrielly ; Valerio, Hellen P. ; Massafera, Mariana P. ; Reis, Lorenna R. ; Coelho, Fernando R. ; Di Mascio, Paolo ; Ronsein, Graziella E.

• Document type: Journal article
• Source: Journal of Leukocyte Biology; v. N/A, p. 12-pg., 2024-12-05.

Abstract

Neutrophils are the innate immune system's first line of defense, and their storage organelles are essential to their function. The storage organelles are divided into 3 different granule types named azurophilic, specific, and gelatinase granules, besides a fourth component called secretory vesicles. The isolation of neutrophil's granules is challenging, and the existing procedures rely on large sample volumes, about 400 mL of peripheral blood, precluding the use of multiple biological and technical replicates. Therefore, the aim of this study was to develop a miniaturized isolation of neutrophil granules method, using biochemical assays, mass spectrometry-based proteomics and a machine learning approach to investigate the protein content of these organelles. Neutrophils were isolated from 40 mL of blood collected from 3 apparently healthy volunteers and disrupted using nitrogen cavitation; the organelles were fractionated with a discontinuous 3-layer Percoll density gradient. The method was proven successful and allowed for a reasonable separation and enrichment of neutrophil's storage organelles using a gradient approximately 37 times smaller than the methods described in the literature. Moreover, mass spectrometry-based proteomics identified 368 proteins in at least 3 of the 5 analyzed samples, and using a machine learning strategy aligned with markers from the literature, the localization of 50 proteins was predicted with confidence. When using markers determined within our dataset by a clusterization tool, the localization of 348 proteins was confidently determined. Importantly, this study was the first to investigate the proteome of neutrophil granules using technical and biological replicates, creating a reliable database for further studies. The miniaturized isolation of neutrophil granules method allowed the enrichment and characterization of neutrophil granules content using 30 times less cells than previous methods. (AU)

• FAPESP's process: 23/00995-4 - Development of metrics of quantitative proteomics capable of capturing the functional and compositional complexity of high-density lipoprotein
• Grantee: Graziella Eliza Ronsein
• Support Opportunities: Regular Research Grants
• FAPESP's process: 16/00696-3 - Proteomic as a tool to understand HDL function and composition
• Grantee: Graziella Eliza Ronsein
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 12/12663-1 - Singlet molecular oxygen and peroxides in chemical biology
• Grantee: Paolo Di Mascio
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC