Updated project metadata. Alternative open reading frames (AltORFs) are unannotated sequences in genome that encode novel peptides or proteins named alternative proteins (AltProts). Although ribosome profiling and bioinformatics predict a large number of AltProts, mass spectrometry (MS) as the only direct way of identification, is hampered by the short lengths and relative low abundance of AltProts. Here we report an approach based on size-exclusion chromatography (SEC) for simultaneous enrichment and fractionation of AltProts from mouse livers. With this strategy we compared and discovered 89 novel AltProts between embryonic and adult mouse livers and will extend our optimized workflow in various disease models in future study.