On 2014 Aug 08, Pavel Baranov commented:

The number of protein coding ORFs in the human transcriptome is likely to vary greatly depending on what we set as the minimal lenghth of such ORFs. Counting genes in the genomes may turn to be unproductive, the term gene is in an existential crisis.

On 2014 Jul 30, Christopher Southan commented:

As was pointed out in http://www.ncbi.nlm.nih.gov/pubmed/15174140 it is incumbent on those proposing novel/alternative ORFs, on whatever evidence basis, to try to collaboratively progress these into "mainstream" protein entries (i.e. as fully expert annoted RefSeq and Swiss-Prot entries). As hitherto, this requires at least the submission of independantly sequenced transcripts with a CDS to the INSDC. UniProt can also then hook-in corroborative MS data via PRIDE (but promotion to Swiss-Prot may require a supporting PubMed citation). As XR points out, bringing verified AltORFs from data limbo land into the cannonical protein fold may need adaptions and expansions to the RefSeq and UniProt criteria (e.g. inclusion of specific antibody data, mass-spec SRM, the promotion of TPA entries ect. See relevant comments at http://cdsouthan.blogspot.se/2011/06/so-is-it-or-isnt-it-protein.html

On 2014 Jul 15, Xavier Roucou commented:

Among several significant contributions in this work, the discovery of 44 novel protein-coding open reading frames (ORFs) illustrates the complexity of the human proteome. Recently, we reported the discovery of 83,886 previously undescribed ORFs termed alternative ORFs (AltORFs) Vanderperre B, 2013. AltORFs are defined as ORFs present in the transcriptome that are different from annotated ORFs. We detected 1,259 proteins translated from AltORFs in human biological samples Vanderperre B, 2013. While the role and importance of this “alternative proteome” will require substantial further validation, there can be no doubt that a comprehensive description of the human proteome must include the distinct possibility of a vastly greater number of functional proteins than has been traditionally considered. Given the existence of the alternative proteome, it is not surprising that Kim et al. found that nearly 50% of the 35 million MS/MS spectra of human proteins did not match proteins in the NCBI’s RefSeq human protein sequence database. In an attempt to identify these novel proteins, the authors translated the human reference genome, RefSeq transcript sequences, non-coding RNAs, and pseudogenes. Among the 193 newly identified proteins, 44 were translated from novel uORFs, ORFs located in an alternate reading frame within coding regions of annotated genes, or ORFs located in 3’-UTRs. The astonishing failure to have detected the alternative proteome years ago results from the fact that MS-based proteomic methods rely on existing protein sequence databases that are far from complete and therefore do not allow the assignment of all MS/MS spectra. Recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals Ingolia NT, 2011 Lee S, 2012 Michel AM, 2012, and these alternative proteins should have been detected. In order to better define the human proteome, we generated a new database of alternative ORFs (AltORFs) present in NCBI’s RefSeq human mRNA sequence database. AltORFs overlap the annotated or reference protein coding ORF (RefORF) in an alternate reading frame, are located in the 5′- and 3′-UTR regions of an mRNA, or partially overlap with both the RefORF and an UTR region. This approach led to the discovery of 83,886 unique AltORFs with a minimum size of 40 codons Vanderperre B, 2013. The majority of mRNAs (87%) have at least one predicted AltORF, with an average of 3.88 AltORFs per mRNA. Additionally, the evolutionary conservation of many of these reading frames suggests functional importance. These AltORFs were translated in silico and included in an alternative protein database we used to interpret unmatched MS/MS spectra. So far, we and others have identified nearly 1300 alternative proteins in different human cell lines and tissues Vanderperre B, 2013, Klemke M, 2001 Oyama M, 2004 Vanderperre B, 2011 Bergeron D, 2013 Slavoff SA, 2013 Menschaert G, 2013, including certain of the 44 new proteins mentioned in the Kim et al. study: the alternative protein translated from the AltORFs mapping to the 5’-UTR of the SLC35A4 gene (or AltSLC35A4), was detected in Hela cells and lung tissue; the AltC11orf48 was detected in Hela cells, colon, lung and ovary tissues; and the AltCHTF8 was detected in Hela cells Vanderperre B, 2013. Twenty four of the 44 novel ORFs detected by Kim et al. were, in fact, already present in our AltORF database, and 9 of the 44 proteins translated from these novel ORFs were previously detected: AltASNSD1, AltSLC35A4, AltMKKS, AltSMCR7L, AltCHTF8, AltRPP14, AltSF1, AltC110rf48, AltHNRNPUL12. In this sense, Kim et al.`s study strongly supports the existence of the alternative proteome. Clearly, the alternative proteins detected by Kim et al. and by our team are the proverbial tip of the iceberg. A full map of the human proteome is thus still years away, and will require several important changes in our current thinking concerning the proteome and the concept that each mature mRNA only codes for one protein.

On 2014 Jun 22, Pavel Baranov commented:

This is a wonderful work and the resource is very useful. Regrettably references to a "novel" protein coding transcript in C11ORF48 locus are missing. Its protein coding properties were identified with ribosome profiling and analyzed using phylogenetic approaches, see Michel AM, 2012. The corresponding N-terminal peptide was detected with mass spectrometry even earlier, see Oyama M, 2007.

On 2014 Jun 22, Pavel Baranov commented:

This is a wonderful work and the resource is very useful. Regrettably references to a "novel" protein coding transcript in C11ORF48 locus are missing. Its protein coding properties were identified with ribosome profiling and analyzed using phylogenetic approaches, see Michel AM, 2012. The corresponding N-terminal peptide was detected with mass spectrometry even earlier, see Oyama M, 2007.

On 2014 Jul 15, Xavier Roucou commented:

Among several significant contributions in this work, the discovery of 44 novel protein-coding open reading frames (ORFs) illustrates the complexity of the human proteome. Recently, we reported the discovery of 83,886 previously undescribed ORFs termed alternative ORFs (AltORFs) Vanderperre B, 2013. AltORFs are defined as ORFs present in the transcriptome that are different from annotated ORFs. We detected 1,259 proteins translated from AltORFs in human biological samples Vanderperre B, 2013. While the role and importance of this “alternative proteome” will require substantial further validation, there can be no doubt that a comprehensive description of the human proteome must include the distinct possibility of a vastly greater number of functional proteins than has been traditionally considered. Given the existence of the alternative proteome, it is not surprising that Kim et al. found that nearly 50% of the 35 million MS/MS spectra of human proteins did not match proteins in the NCBI’s RefSeq human protein sequence database. In an attempt to identify these novel proteins, the authors translated the human reference genome, RefSeq transcript sequences, non-coding RNAs, and pseudogenes. Among the 193 newly identified proteins, 44 were translated from novel uORFs, ORFs located in an alternate reading frame within coding regions of annotated genes, or ORFs located in 3’-UTRs. The astonishing failure to have detected the alternative proteome years ago results from the fact that MS-based proteomic methods rely on existing protein sequence databases that are far from complete and therefore do not allow the assignment of all MS/MS spectra. Recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals Ingolia NT, 2011 Lee S, 2012 Michel AM, 2012, and these alternative proteins should have been detected. In order to better define the human proteome, we generated a new database of alternative ORFs (AltORFs) present in NCBI’s RefSeq human mRNA sequence database. AltORFs overlap the annotated or reference protein coding ORF (RefORF) in an alternate reading frame, are located in the 5′- and 3′-UTR regions of an mRNA, or partially overlap with both the RefORF and an UTR region. This approach led to the discovery of 83,886 unique AltORFs with a minimum size of 40 codons Vanderperre B, 2013. The majority of mRNAs (87%) have at least one predicted AltORF, with an average of 3.88 AltORFs per mRNA. Additionally, the evolutionary conservation of many of these reading frames suggests functional importance. These AltORFs were translated in silico and included in an alternative protein database we used to interpret unmatched MS/MS spectra. So far, we and others have identified nearly 1300 alternative proteins in different human cell lines and tissues Vanderperre B, 2013, Klemke M, 2001 Oyama M, 2004 Vanderperre B, 2011 Bergeron D, 2013 Slavoff SA, 2013 Menschaert G, 2013, including certain of the 44 new proteins mentioned in the Kim et al. study: the alternative protein translated from the AltORFs mapping to the 5’-UTR of the SLC35A4 gene (or AltSLC35A4), was detected in Hela cells and lung tissue; the AltC11orf48 was detected in Hela cells, colon, lung and ovary tissues; and the AltCHTF8 was detected in Hela cells Vanderperre B, 2013. Twenty four of the 44 novel ORFs detected by Kim et al. were, in fact, already present in our AltORF database, and 9 of the 44 proteins translated from these novel ORFs were previously detected: AltASNSD1, AltSLC35A4, AltMKKS, AltSMCR7L, AltCHTF8, AltRPP14, AltSF1, AltC110rf48, AltHNRNPUL12. In this sense, Kim et al.`s study strongly supports the existence of the alternative proteome. Clearly, the alternative proteins detected by Kim et al. and by our team are the proverbial tip of the iceberg. A full map of the human proteome is thus still years away, and will require several important changes in our current thinking concerning the proteome and the concept that each mature mRNA only codes for one protein.