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Related Experiment Video

Updated: Jan 3, 2026

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames
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Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing.

Laura M Agosto1,2,3, Matthew R Gazzara2,4, Caleb M Radens2,5

  • 1Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Genome Research
|November 16, 2019
PubMed
Summary
This summary is machine-generated.

This study reveals that many alternatively spliced messenger RNAs (mRNAs) are translated into proteins. A new workflow enhances the detection of these splicing-derived proteoforms in large-scale proteomic experiments.

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Area of Science:

  • Molecular Biology
  • Proteomics
  • Bioinformatics

Background:

  • Alternative pre-messenger RNA (mRNA) splicing is a key mechanism for generating proteome diversity.
  • The extent to which alternatively spliced mRNA isoforms are translated into functional proteins remains largely unquantified and debated.

Purpose of the Study:

  • To accurately quantify the translation of alternatively spliced mRNAs into proteins.
  • To develop and validate an improved workflow for detecting splicing-derived proteoforms in large-scale proteomic studies.

Main Methods:

  • Utilized high-throughput RNA sequencing (RNA-seq) and mass spectrometry (MS)-based proteomics.
  • Optimized cell fractionation and sample processing for enhanced proteome coverage and protein quantitation.
  • Generated a custom peptide database using MAJIQ algorithm for splice junction analysis, integrated with SWISS-PROT and RefSeq databases.

Main Results:

  • Identified peptide evidence for 554 alternate proteoforms from 274 genes, a 28% increase compared to using SWISS-PROT alone.
  • Successfully tracked transcriptome and proteome changes induced by T-cell stimulation, including protein subcellular localization shifts.
  • Demonstrated that generic databases underestimate the number of translated spliced mRNA isoforms.

Conclusions:

  • Confirms that a significant number of alternatively spliced mRNA isoforms are translated into proteins.
  • Provides a robust workflow that enhances the detection and quantitation of proteoforms in large-scale proteomic experiments.
  • Highlights the underestimation of translated splice variants when using standard proteomic databases.