The data presented here indicate an average of 3.8 AltORFs per human mRNA with a median length of 57 amino acids. Using this database, 1259 human alternative proteins were detected by mass spectrometry in the present study, 3 of which were previously detected. This result strongly supports the hypothesis that the complexity of the proteome has been underestimated and alternative translation initiation already well characterized in viruses cannot be ignored in humans. 
Importantly, evolutionary conservation of alternative proteins between vertebrates and invertebrate implies that these proteins have significant biological functions. It is very likely that similar to proteins translated from canonical ORFs, alternative proteins display a wide variety of biological functions. This is suggested by the great diversity of subcellular localizations that we observed in our fluorescence microscopy experiments, and by the fact that a growing number of important Fulvestrant company functions are attributed to small proteins and peptides. The polycistronic nature of AltORF encoding mRNAs can potentially lead to intriguing functional interplays between reference and alternative proteins, such as direct interaction between the reference and alternative proteins. There is also evidence that an upstream ORF not only regulates the expression of a downstream RefORF by interfering in cis with canonical AUG recognition by scanning ribosomes, but also reduces the translational efficiency of the RefORF in trans. AltORFs translation products could also be of particular importance during the thymal selection of T lymphocytes, serving as “cryptic T-cell epitopes”. In some cases, this has been shown to lead to the selection of lymphocytes with antiviral or antitumor activities. Our databases of AltORFs will be useful to identify genes containing multiple protein coding ORFs and to unravel their functions. This is particularly important in experimental settings where gene expression studies could result in the expression or down regulation of a reference protein and an unnoticed alternative protein, leading to confounding results. Another striking example is the co-expression of therapeutic transgenes and their associated alternative proteins, which elicit a cytotoxic T lymphocyte response. Thus, transgene sequences should be carefully examined for possible AltORFs to decrease potential adverse immune responses during therapeutic gene transfer. We also propose that proteins recalcitrant to mass spectrometry identification or proteins with no sequence homology with the conventional proteome identified in large-scale cDNAs screens should be revisited with the AltORFs databases. Additionally, large fundamental and clinical proteomic studies using organs and tissues would likely benefit from the AltORFs database to achieve complete catalogs of proteins in different tissues. The presence of a large LY2109761 fraction of alternative proteins in plasma and serum is particularly interesting as there is a constant need for biomarkers to identify a variety of disorders at an early stage. The reason why so many alternative proteins are secreted is currently unknown. We did not find any enrichment for classical export signal peptides, and their secretion mechanism remains to be investigated. As for any databases, the AltORFs database has some limitations. Although AUG remains the main translation initiation site, recent ribosome profiling studies clearly indicate the use of non-AUG start sites. Yet, we did not take into account nonAUG initiation sites as an accurate prediction method for such functional translation start sites is not yet available.