Validating Amino Acid Variants in Proteogenomics Using Sequence Coverage by Multiple Reads.

Area of Science:

• Proteomics
• Genomics
• Biochemistry

Background:

• Mass spectrometry-based proteome analysis relies on matching peptide mass spectra to genomic sequences.
• Current proteogenomic studies often lack reliability in identifying peptide variants.

Purpose of the Study:

• To propose a new method for interpreting shotgun proteomics data, specifically in data-dependent acquisition mode.
• To enhance the reliability of peptide variant identification by treating proteomics data similarly to nucleic acid sequencing.

Main Methods:

• Interpreting shotgun proteomics results as protein sequence coverage by multiple reads.
• Utilizing overlapping distinct peptides (from miscleaved and properly cleaved tryptic peptides, or parallel digestion with multiple proteases) to confirm amino acid residues.
• Analyzing publicly available multiprotease datasets and HEK-293 cell line digests (trypsin, LysC, GluC).

Main Results:

• Up to 30% of the whole proteome was covered by tryptic peptides.
• Up to 7% of the proteome was covered by twofold or more.
• Proteogenomic analysis of HEK-293 cells identified 36 single amino acid variants, with 7 supported by multiple reads.

Conclusions:

• The proposed method increases confidence in amino acid residue identification through overlapping peptide analysis.
• This approach significantly lowers the false discovery rate for peptide variant identification in proteogenomics.
• The study demonstrates the utility of multiprotease digestion and overlapping peptide analysis for robust proteogenomic discoveries.