Chromosomal proteins: Placental proteome in the C-HPP

Chromosome-Centric Human Proteome Project

The Chromosome-Centric Human Proteome Project (C-HPP) was established to map all human proteins that are annotated by genes on each chromosome. In the initial stages, the goals are to identify at least one representative protein encoded by each of the 20,059 human genes, then organize the data in accordance with the chromosomal structures. To achieve these difficult targets, 25 working groups were set up around the world and one of these has just reported its findings.

Within each working group, established proteins will be confirmed, new proteins will be identified, including their major post-translational modifications, and missing proteins which are predicted but as yet unseen in practice will be found, so that all of the human genes are represented in the final data set.

A collaboration in Korea led by Young-Ki Paik from Yonsei University, Seoul, chose to work with the human placenta. This organ is known to contain the greatest number of genes of any organ in the human body but no large-scale genome-side proteome analyses have been carried out to date. So, the team established a set of working procedures for the placenta which could be extended to any clinical samples examined within the C-HPP.

Placenta processing

Placental tissues were collected from uncomplicated pregnancies and subjected to a number of fractionation techniques to target different types of proteins. For large-scale profiling, the proteins present were digested with trypsin and fractionated by HILIC, strong cation exchange or OFFGEL electrophoresis, a commercial isoelectric focussing technique.

Alternatively, the glycoproteins and phosphoproteins were analysed following separation by multilectin affinity chromatography and titanium dioxide enrichment, respectively. Finally, the proteins in placentas from normal and preeclampsia pregnancies were labelled with the tandem mass tag and mixed before OFFGEL electrophoresis to compare their abundances and highlight those involved with the disease.

All of the fractions from these techniques were analysed by LC-tandem-MS on a high-resolution mass spectrometer operating in collision-induced dissociation (CID), high-energy CID and electron-transfer dissociation modes. The proteins were identified using UniProt and the IPI human sequence database.

Proteins for chromosomes

Overall, 4239 unique proteins were confidently identified in the placental tissue covering more than 21% of the total predicted human proteins, based on one per gene. They included 33 missing proteins. This number is the highest number of proteins reported to date from this tissue. Within the protein pool were 219 unique N-linked glycopeptides and 592 unique phosphopeptides.

The proteins were assigned to the human chromosomes and the distribution was found to be proportional to the gene count distribution of each chromosome. The research team were primarily interested in chromosome 13, for which 66 placental proteins were found. That makes a total of 141 proteins for this chromosome when they were combined with those found by a second Korean team working on chromosome 11 which examined brain tissue. The results also confirmed the 14 genes that are known to be up-regulated in the placenta by the presence of their associated proteins.

A total of 1331 proteins were also quantified with high confidence and 28 were differentially expressed in the preeclampsia samples, suggesting some involvement with the condition. However, the identities of the proteins were largely different from the 21 that were found by a parallel label-free approach which the team also carried out. They attributed the variations to different sensitivities of the labelled and unlabelled proteins during quantification.

The research group recommended that future studies should examine the biological roles of the placental proteins, especially those involved in preeclampsia. This could lead to novel drug targets, molecular diagnostics and therapeutic monitoring, which are some of the outcomes expected from C-HPP.

The raw data from the study will be deposited in a public database for use by all of the working groups and the successful experimental protocol will help in the development of a standardised platform for all of the C-HPP working groups.