Identification and Validation of Human Missing Proteins and Peptides in Public Proteome Databases: Data Mining...

Area of Science:

• Proteomics
• Genomics
• Human Proteome Project

Background:

• The Chromosome-Centric Human Proteome Project (C-HPP) aims to complete the human proteome.
• Significant numbers of missing (2579) and uncertain (572) proteins remain in neXtProt (2017-1).
• Low abundance and limited unique tryptic peptides are key challenges in detecting missing proteins.

Purpose of the Study:

• To develop and implement a pipeline for analyzing, identifying, and validating human missing and uncertain proteins.
• To investigate the expression patterns and challenges associated with missing proteins.
• To leverage open-access transcriptomics and proteomics databases for protein discovery.

Main Methods:

• Analysis of RNA expression patterns in the Human Protein Atlas.
• Utilizing open-access mass spectrometry (MS)-based proteomics databases (PA, GPM).
• Searching for predicted unique tryptic peptides and validating with native spectra against SRMAtlas synthetic counterparts.

Main Results:

• 28% of missing proteins showed no RNA expression; 21% were tissue-enriched.
• 95% of missing proteins had low RNA expression (0-10 TPM), indicating low abundance challenges.
• Identified 402 missing and 19 uncertain proteins with ≥2 unique peptides; validated 41 missing proteins using ≥2 proteotypic peptides.

Conclusions:

• The developed pipeline is effective for identifying and validating missing and uncertain proteins.
• Low abundance and limited unique peptides are significant hurdles for missing protein detection.
• Targeting tissue-enriched proteins and exploring uncommon tissues are crucial strategies for future proteomic studies.