title: Discovery of colorectal cancer biomarker candidates by membrane proteomic analysis and subsequent verification using selected reaction monitoring (SRM) and tissue microarray (TMA) analysis.
abstract: Recent advances in quantitative proteomic technology have enabled the large-scale validation of biomarkers. We here performed a quantitative proteomic analysis of membrane fractions from colorectal cancer tissue to discover biomarker candidates, and then extensively validated the candidate proteins identified. A total of 5566 proteins were identified in six tissue samples, each of which was obtained from polyps and cancer with and without metastasis. GO cellular component analysis predicted that 3087 of these proteins were membrane proteins, whereas TMHMM algorithm predicted that 1567 proteins had a transmembrane domain. Differences were observed in the expression of 159 membrane proteins and 55 extracellular proteins between polyps and cancer without metastasis, while the expression of 32 membrane proteins and 17 extracellular proteins differed between cancer with and without metastasis. A total of 105 of these biomarker candidates were quantitated using selected (or multiple) reaction monitoring (SRM/MRM) with stable synthetic isotope-labeled peptides as an internal control. The results obtained revealed differences in the expression of 69 of these proteins, and this was subsequently verified in an independent set of patient samples (polyps (n = 10), cancer without metastasis (n = 10), cancer with metastasis (n = 10)). Significant differences were observed in the expression of 44 of these proteins, including ITGA5, GPRC5A, PDGFRB, and TFRC, which have already been shown to be overexpressed in colorectal cancer, as well as proteins with unknown function, such as C8orf55. The expression of C8orf55 was also shown to be high not only in colorectal cancer, but also in several cancer tissues using a multicancer tissue microarray, which included 1150 cores from 14 cancer tissues. This is the largest verification study of biomarker candidate membrane proteins to date; our methods for biomarker discovery and subsequent validation using SRM/MRM will contribute to the identification of useful biomarker candidates for various cancers. Data are available via ProteomeXchange with identifier PXD000851.
authors.affiliation: From the ‡Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka, Japan;
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kume
authors.affiliation: From the ‡Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka, Japan;
authors.forename: Satoshi
authors.initials: S
authors.lastname: Muraoka
authors.affiliation: From the ‡Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka, Japan;
authors.forename: Takahisa
authors.initials: T
authors.lastname: Kuga
authors.affiliation: From the ‡Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka, Japan;
authors.forename: Jun
authors.initials: J
authors.lastname: Adachi
authors.affiliation: From the ‡Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka, Japan;
authors.forename: Ryohei
authors.initials: R
authors.lastname: Narumi
authors.affiliation: From the ‡Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka, Japan;
authors.forename: Shio
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: From the ‡Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka, Japan;
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Kuwano
authors.affiliation: §Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science, Kanagawa, Japan;
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kodera
authors.affiliation: ¶Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan; ‖Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan;
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsushita
authors.affiliation: **Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan;
authors.forename: Junya
authors.initials: J
authors.lastname: Fukuoka
authors.affiliation: ‡‡Institute for Advanced Biosciences, Keio University, Yamagata, Japan;
authors.forename: Takeshi
authors.initials: T
authors.lastname: Masuda
authors.affiliation: §§Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan;
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
authors.affiliation: ¶¶Department of Frontier Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Hisahiro
authors.initials: H
authors.lastname: Matsubara
authors.affiliation: ¶Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan; ‖Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan;
authors.forename: Fumio
authors.initials: F
authors.lastname: Nomura
authors.affiliation: From the ‡Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka, Japan; tomonaga@nibio.go.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tomonaga
mesh_terms: D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000302:isolation & purification; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D040901:Proteomics; D046888:Tissue Array Analysis
chemical_list: D014408:Biomarkers, Tumor; D008565:Membrane Proteins; D009363:Neoplasm Proteins