An important goal of the Human Proteome Organization (HUPO) Chromosome-Centric Human Proteome Project (C-HPP) is to correctly define the number of canonical protein encoded by their cognate open reading frames on each chromosome in the human genome. When identified with high confidence of protein evidence (PE) such proteins are termed PE1 proteins in the online database resource, neXtProt. However, proteins that have not been identified unequivocally at the protein level, but that have other evidence suggestive of their existence (PE2 – 4), are termed missing proteins (MPs). The numbers of MPs have been reduced from 5,511 in 2012 to 2,186 in 2018 (neXtProt 2018-01-17 release). Whereas the annotation of the HPP is significant, the ‘parts list’ alone does not inform function. Indeed, 1,937 proteins representing ~10% of the human proteome have no function annotated either from experimental characterization or predicted by homology to other proteins. Specifically, these 1,937 ‘dark proteins’ of the so-called dark proteome are composed of 1,260 functionally uncharacterized but identified PE1 proteins, designated as uPE1, plus 677 MPs from categories PE2 – PE4, which also have no known or predicted function and are termed uMPs. At the HUPO-2017 Annual Meeting, the C-HPP officially adopted the uPE1 pilot initiative, with 14 participating international teams later committing to demonstrate the feasibility of the functional characterization of large numbers of dark proteins, starting first with 50 uPE1 proteins, in a stepwise chromosome-centric organizational manner. The second aim of the feasibility phase of the ‘neXt-CP50’ initiative to determine functions of uPE1 proteins is to utilize a variety of approaches and workflows according to individual team expertise, interest, and resources so as to enable the C-HPP to recommend experimentally proven workflows to the proteome community within 3 years. The results from this pilot will not only be the cornerstone of a larger characterization initiative, but also enhance understanding of the human proteome and integrated cellular networks for the discovery of new mechanisms of pathology, mechanistically informative biomarkers, and rational drug targets.

As a starting point of the Chromosome-Centric Human Proteome Project (C-HPP), we established strategies of genome-wide proteomic analysis, including protein identification, quantitation of disease-specific proteins, and assessment of post-translational modifications, using paired human placental tissues from healthy and preeclampsia patients. This analysis resulted in identification of 4239 unique proteins with high confidence (two or more unique peptides with a false discovery rate less than 1%), covering 21% of approximately 20, 059 (Ensembl v69, Oct 2012) human proteins, among which 28 proteins exhibited differentially expressed preeclampsia-specific proteins. When these proteins are assigned to all human chromosomes, the pattern of the newly identified placental protein population is proportional to that of the gene count distribution of each chromosome. We also identified 219 unique N-linked glycopeptides, 592 unique phosphopeptides, and 66 chromosome 13-specific proteins. In particular, protein evidence of 14 genes previously known to be specifically up-regulated in human placenta was verified by mass spectrometry. With respect to the functional implication of these proteins, 38 proteins were found to be involved in regulatory factor biosynthesis or the immune system in the placenta, but the molecular mechanism of these proteins during pregnancy warrants further investigation. As far as we know, this work produced the highest number of proteins identified in the placenta and will be useful for annotating and mapping all proteins encoded in the human genome.

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