Table 1:

Features and characteristics of methods to detect non-canonical ORF translation

	Molecule detected	Digestion step?	Target size of analyte	Number of annotated CDSs detected	Number of ORFs detected	Strengths	Weaknesses
Ribo-seq	RNA bound within ribosomes	RNase and DNase	28 – 30 nt	10,000 – 13,000	2,000 – 200,000	-Genome-wide -No bias due to trypsin -Detects small and large CDSs -Nucleotide-level precision -Defines exact reading frame of ORF	-Does not detect proteins directly -Cannot detect PTMs -Cannot inform post-translational protein regulation -Analysis pipelines may be discordant
LC-MS/MS	Tryptic peptides	Trypsin	8 – 25 amino acids	9.000 – 11.000	10s to 100s	-Direct protein detection -Informs protein abundance -May detect PTMs -Proteome-wide	-High false-positive rate without Ribo-seq -Biased against small proteins -Trypsin may bias protein representation -Does not provide nucleotide-level precision
HLA Immuno-peptidomics	HLA-presented peptide antigens	None	8 – 12 amino acids	8,000 – 10,000	1000 – 5000	-Direct protein detection -Enrichment for low-abundance, strong binders -Proteome-wide -Can detect unstable translations -Does not require tryptic sites	-Does not inform protein stability -Does not indicate intracellular abundance -HLA allele expression limits peptide representation -Does not provide nucleotide-level precision