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Next‐generation transcriptome sequencing is increasingly integrated with MS to enhance MS‐based protein and peptide identification. Recently, a breakthrough in transcriptome analysis was achieved with the development of ribosome profiling (ribo‐seq). This technology is based on the deep sequencing of ribosome‐protected mRNA fragments, thereby enabling the direct observation of in vivo protein synthesis at the transcript level. In order to explore the impact of a ribo‐seq‐derived protein sequence search space on MS/MS spectrum identification, we performed a comprehensive proteome study on a human cancer cell line, using both shotgun and N‐terminal proteomics, next to ribosome profiling, which was used to delineate (alternative) translational reading frames. By including protein‐level evidence of sample‐specific genetic variation and alternative translation, this strategy improved the identification score …