Global detection of human variants and isoforms by deep proteome sequencing.

Area of Science:

• Proteomics
• Molecular Biology
• Genomics

Background:

• Shotgun proteomics typically identifies proteins using limited peptide sequences, failing to distinguish variants and isoforms.
• Deeper proteome sequencing is essential for comprehensive discovery of protein variants and isoforms.

Purpose of the Study:

• To perform deep proteome sequencing for global discovery of protein isoforms.
• To investigate the translation of nonsynonymous variants and alternatively spliced isoforms.
• To establish a dataset for proteoform discovery.

Main Methods:

• Utilized six human cell lines, six proteases, deep fractionation, and three tandem mass spectrometry fragmentation methods.
• Identified over a million unique peptides across 17,717 protein groups.
• Achieved a median sequence coverage of approximately 80%.

Main Results:

• Provided evidence for the translation of most nonsynonymous variants through comparison with RNA expression data.
• Observed comparable detection rates for peptides representing both constitutive and alternative splicing.
• Hypothesized that undetected variants may result from mutation-induced protein instability.

Conclusions:

• The study generated a valuable dataset for proteoform discovery.
• Demonstrated direct evidence that most frame-preserving alternatively spliced isoforms are translated.
• Highlights the capability of deep proteome sequencing to uncover protein diversity.