A proteomics sample metadata representation for multiomics integration and big data analysis

The amount of public proteomics data is rapidly increasing but there is no standardized format to describe the sample metadata and their relationship with the dataset files in a way that fully supports their understanding or reanalysis. Here we propose to develop the transcriptomics data format MAGE-TAB into a standard representation for proteomics sample metadata. We implement MAGE-TAB-Proteomics in a crowdsourcing project to manually curate over 200 public datasets. We also describe tools and libraries to validate and submit sample metadata-related information to the PRIDE repository. We expect that these developments will improve the reproducibility and facilitate the reanalysis and integration of public proteomics datasets.publishedVersio

Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration

The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells. From the 2669 unique phosphorylation sites identified, 77 and 43 were differentially phosphorylated in the tumor cells and endothelial cells, respectively. The receptor tyrosine kinase ephrin type A receptor 2 (EPHA2) exhibited decreased Tyr(772) phosphorylation in the cancer cells upon endothelial contact. Knockdown of EPHA2 increased adhesion of the breast cancer cells to human umbilical vein endothelial cells (HUVECs) and their transendothelial migration in coculture cell assays, as well as early-stage lung colonization in vivo. EPHA2-mediated inhibition of transendothelial migration of breast cancer cells depended on interaction with the ligand ephrinA1 on HUVECs and phosphorylation of EPHA2-Tyr(772). When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr(772) was rapidly dephosphorylated after ephrinA1 stimulation specifically in cells targeting the lung. Knockdown of the phosphatase LMW-PTP reduced adhesion and transendothelial migration of the breast cancer cells. Overall, cell-specific phosphoproteomic analysis provides a bidirectional map of contact-initiated signaling between tumor and endothelial cells that can be further investigated to identify mechanisms controlling the transendothelial cell migration of cancer cells

Hunting for the elusive target antigen in gestational alloimmune liver disease (GALD).

The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in Escherichia coli using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor

Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.Funding: ProteoRed, PRB3 is supported by grant PT17/0019/0001, of the PE I+D+i 2013-2016, funded by ISCIII and ERD