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J Proteome Res. 2017 Dec 1;16(12):4455-4467. doi: 10.1021/acs.jproteome.7b00146. Epub 2017 Nov 1.

Systematic Proteogenomic Approach To Exploring a Novel Function for NHERF1 in Human Reproductive Disorder: Lessons for Exploring Missing Proteins.

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Yonsei Proteome Research Center, Yonsei University , Seoul 03722, South Korea.
Department of Integrated OMICS for Biomedical Science, Yonsei University , Seoul 03722, South Korea.
Department of Obstetrics and Gynecology, CHA Bundang Medical Center, CHA University , Seongnam 13496, South Korea.
CHA Biotech Co., Ltd. , Seongnam 13488, South Korea.
Department of Biochemistry, CHA University , Seongnam 13488, South Korea.
Department of Medicine, School of Medicine, Konkuk University , Seoul 143701, South Korea.
Center for Computational Medicine and Bioinformatics, University of Michigan , Ann Arbor, Michigan 48109, United States.
Department of Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University , Seoul 03722, South Korea.


One of the major goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to fill the knowledge gaps between human genomic information and the corresponding proteomic information. These gaps are due to "missing" proteins (MPs)-predicted proteins with insufficient evidence from mass spectrometry (MS), biochemical, structural, or antibody analyses-that currently account for 2579 of the 19587 predicted human proteins (neXtProt, 2017-01). We address some of the lessons learned from the inconsistent annotations of missing proteins in databases (DB) and demonstrate a systematic proteogenomic approach designed to explore a potential new function of a known protein. To illustrate a cautious and strategic approach for characterization of novel function in vitro and in vivo, we present the case of Na(+)/H(+) exchange regulatory cofactor 1 (NHERF1/SLC9A3R1, located at chromosome 17q25.1; hereafter NHERF1), which was mistakenly labeled as an MP in one DB (Global Proteome Machine Database; GPMDB, 2011-09 release) but was well known in another public DB and in the literature. As a first step, NHERF1 was determined by MS and immunoblotting for its molecular identity. We next investigated the potential new function of NHERF1 by carrying out the quantitative MS profiling of placental trophoblasts (PXD004723) and functional study of cytotrophoblast JEG-3 cells. We found that NHERF1 was associated with trophoblast differentiation and motility. To validate this newly found cellular function of NHERF1, we used the Caenorhabditis elegans mutant of nrfl-1 (a nematode ortholog of NHERF1), which exhibits a protruding vulva (Pvl) and egg-laying-defective phenotype, and performed genetic complementation work. The nrfl-1 mutant was almost fully rescued by the transfection of the recombinant transgenic construct that contained human NHERF1. These results suggest that NHERF1 could have a previously unknown function in pregnancy and in the development of human embryos. Our study outlines a stepwise experimental platform to explore new functions of ambiguously denoted candidate proteins and scrutinizes the mandated DB search for the selection of MPs to study in the future.


C-HPP; NHERF1; SLC9A3R1; missing protein; preeclampsia; proteogenomics

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