Validating Missing Proteins in Human Sperm Cells by Targeted Mass-Spectrometry- and Antibody-based Methods

Area of Science:

• Proteomics
• Human reproductive biology
• Chromosomal protein analysis

Background:

• The neXt50 challenge aims to identify 50 missing proteins (MPs) per chromosome.
• Human sperm proteome analysis is crucial for understanding reproductive health.
• Identifying MPs in sperm contributes to the comprehensive Human Protein Atlas.

Purpose of the Study:

• To identify 38 targeted missing proteins (MPs) on chromosomes 2 and 14 in human sperm.
• To contribute to the neXt50 challenge and the Human Protein Atlas.
• To explore potential links between identified MPs and human idiopathic infertility.

Main Methods:

• Targeted mass spectrometry using Liquid Chromatography-Parallel Reaction Monitoring (LC-PRM) assays.
• Development of assays targeting 92 proteotypic peptides from 38 selected MPs.
• Analysis of Triton X-100 soluble and insoluble sperm fractions from 15 healthy donors.
• Immunohistochemistry and cytochemistry for validation of selected MPs.

Main Results:

• 12 out of 38 targeted MPs were identified with two or more peptides.
• 3 additional MPs were identified with a single peptide, exhibiting low signal intensity.
• Data are publicly available via ProteomeXchange (PASS01013).
• Eight MPs were validated using immunohistochemistry and cytochemistry.

Conclusions:

• Deep proteomic analysis of human sperm can validate missing proteins.
• This study contributes to the global C-HPP effort to map the human proteome.
• Identified MPs may serve as novel candidates for investigating human idiopathic infertility.