Comparative Proteomic Profiling of Unannotated Microproteins and Alternative Proteins in Human Cell Lines

Ribosome
profiling and mass spectrometry have revealed thousands
of small and alternative open reading frames (sm/alt-ORFs) that are
translated into polypeptides variously termed as microproteins and
alt-proteins in mammalian cells. Some micro-/alt-proteins exhibit
stress-, cell-type-, and/or tissue-specific expression; understanding
this regulated expression will be critical to elucidating their functions.
While differential translation has been inferred by ribosome profiling,
quantitative mass spectrometry-based proteomics is needed for direct
comparison of microprotein and alt-protein expression between samples
and conditions. However, while label-free quantitative proteomics
has been applied to detect stress-dependent expression of bacterial
microproteins, this approach has not yet been demonstrated for analysis
of differential expression of unannotated ORFs in the more complex
human proteome. Here, we present global micro-/alt-protein quantitation
in two human leukemia cell lines, K562 and MOLT4. We identify 12 unannotated
proteins that are differentially expressed in these cell lines. The
expression of six micro/alt-proteins from cDNA was validated biochemically,
and two were found to localize to the nucleus. Thus, we demonstrate
that label-free comparative proteomics enables quantitation of micro-/alt-protein
expression between human cell lines. We anticipate that this workflow
will enable the discovery of regulated sm/alt-ORF products across
many biological conditions in human cells.