Letter pubs.acs.org/jpr
Cite This: J. Proteome Res. XXXX, XXX, XXX−XXX
Identification of Missing Proteins in Human Olfactory Epithelial Tissue by Liquid Chromatography−Tandem Mass Spectrometry Heeyoun Hwang,†,‡,§,¶ Ji Eun Jeong,†,‡,¶ Hyun Kyoung Lee,†,‡ Ki Na Yun,†,∥ Hyun Joo An,‡,§ Bonghee Lee,⊥,# Young-Ki Paik,∇ Tae Seok Jeong,⊗ Gi Taek Yee,⊗ Jin Young Kim,*,† and Jong Shin Yoo*,†,‡ †
Biomedical Omics Research, Korea Basic Science Institute, Cheongju, Korea Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea § Asia-Pacific Glycomics Reference Site, Chungnam National University, Daejeon, Korea ∥ Department of Chemistry, Sogang University, Seoul, Korea ⊥ Department of Anatomy & Cell Biology, Graduate School of Medicine, Gachon University, Incheon, Korea # Center for Genomics and Proteomics & Stem Cell Core Facility, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Korea ∇ Yonsei Proteome Research Center and Department of Integrated OMICS for Biomedical Science, and Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea ⊗ Department of Neurosurgery, Gil Medical Center, Gachon University, Incheon, Korea
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‡
S Supporting Information *
ABSTRACT: We performed proteomic analyses of human olfactory epithelial tissue to identify missing proteins using liquid chromatography−tandem mass spectrometry. Using a next-generation proteomic pipeline with a < 1.0% false discovery rate at the peptide and protein levels, we identified 3731 proteins, among which five were missing proteins (P0C7M7, P46721, P59826, Q658L1, and Q8N434). We validated the identified missing proteins using the corresponding synthetic peptides. No olfactory receptor (OR) proteins were detected in olfactory tissue, suggesting that detection of ORs would be very difficult. We also identified 49 and 50 alternative splicing variants mapped at the neXtProt and GENCODE databases, respectively, and 2000 additional single amino acid variants. This data set is available at the ProteomeXchange consortium via PRIDE repository (PXD010025).
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INTRODUCTION
proteins (68/378) are missing, few studies involving highthroughput liquid chromatography−tandem mass spectrometry (LC−MS/MS) analyses of human olfactory epithelial tissue have been performed.2 A total of 1970 proteins were identified in the membrane-enriched fractions of human nasal epithelium obtained by a noninvasive brushing procedure, with two or more unique peptides detected in one or more samples.3 Without scrutiny of their experimental tandem MS spectra, 20 missing proteins (19 PE2 and 1 PE3) were sorted against the latest version of the neXtProt database. However, that study had an extremely high false discovery rate (FDR, 4.5% at the peptide level), and the raw files were not deposited into any public database. Baker et al. collected 122 717 OR
The human proteome project (HPP) is aimed at identifying novel proteoforms, such as missing proteins (MPs), alternative splicing variants (ASVs) and single amino acid variants (SAAVs), and expanding our understanding of the human proteome.1 In the latest version of the neXtProt database (version 2018− 01−17; https://www.nextprot.org), there are 2186 proteins that have not been detected by proteomic approaches, among which 927 (42.4%) proteins (683 [PE2], 236 [PE3], and 8 [PE4]) have one or more transmembrane domains. To identify these MPs and understand why they are missing, we need to develop approaches focusing on membrane protein enrichment, improving mass spectrometry sensitivity, and preparing specific unusual human samples, such as olfactory epithelial tissue.2 Although most olfactory receptors (OR) (409/411) and ∼18% of other G protein-coupled receptor (GPCR)-family © XXXX American Chemical Society
Special Issue: Human Proteome Project 2018 Received: May 31, 2018 Published: August 16, 2018 A
DOI: 10.1021/acs.jproteome.8b00408 J. Proteome Res. XXXX, XXX, XXX−XXX
Letter
Journal of Proteome Research peptides from public proteomic repositories using a semiautomatic workflow. After filtering reliable peptide spectrum matches, they found 23 OR peptides with a single distinct peptide, but none met the criteria in the HPP guidelines.2
Table 1. Comparison of Proteomic Identification between neXtProt Only and Next-Generation Proteomic Pipeline (nextPP)
MATERIALS AND METHODS To facilitate the identification of membrane proteins and MPs in human olfactory epithelial tissue, which was obtained from the ethmoidal mucosa of the nasal cavity during surgery using a skull base approach (endoscopic endonasal extended transsphenoidal approach) in a patient with anterior skull base meningioma, we performed membrane protein extraction using 4% sodium dodecyl sulfate (SDS) in Tris buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by in-gel digestion, and subjected to basic reverse phase liquid chromatographic (bRPLC) fractionation and nano-LC−MS/MS analyses.4 Briefly, 24 LC−MS/ MS raw files were converted into MS2 files using RawConverter (v. 1.1.0.18, The Scripps Research Institute, La Jolla, CA, USA). The Integrated Proteomic Pipeline (v. 4.1.2.4, The Scripps Research Institute) and ProteinInferencer (v. 1.0, The Scripps Research Institute) were used for proteomic data processing with
