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. 2014 Jun;13(6):1471-84.
doi: 10.1074/mcp.M113.037093. Epub 2014 Mar 31.

Discovery of colorectal cancer biomarker candidates by membrane proteomic analysis and subsequent verification using selected reaction monitoring (SRM) and tissue microarray (TMA) analysis

Hideaki Kume  1 , Satoshi Muraoka  1 , Takahisa Kuga  1 , Jun Adachi  1 , Ryohei Narumi  1 , Shio Watanabe  1 , Masayoshi Kuwano  1 , Yoshio Kodera  2 , Kazuyuki Matsushita  3 , Junya Fukuoka  4 , Takeshi Masuda  5 , Yasushi Ishihama  6 , Hisahiro Matsubara  7 , Fumio Nomura  3 , Takeshi Tomonaga  8
Affiliations

Discovery of colorectal cancer biomarker candidates by membrane proteomic analysis and subsequent verification using selected reaction monitoring (SRM) and tissue microarray (TMA) analysis

Hideaki Kume et al. Mol Cell Proteomics. 2014 Jun.

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.

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Figures

Fig. 1.
Fig. 1.
Outline of the experimental workflow.
Fig. 2.
Fig. 2.
Representative results obtained with iTRAQ and SRM/MRM in the discovery and confirmation steps. A, The iTRAQ and SRM/MRM data for ITGA5, GPRC5A, PDGFRB, and TFRC are shown. P, polyp. C, cancer without metastasis. Cm, cancer with metastasis. Area ratio, the ratio of the peak area of the endogenous peptide to that of the SI peptide. Assays were constructed to measure two distinct peptides per-protein listed in supplemental Table S5 and that the individual assays for each of the two peptides are labeled SRM-1 and SRM-2. B, Western blotting analysis of polyps and cancer with and without metastasis using an anti-ITGA5 antibody.
Fig. 3.
Fig. 3.
Representative results of SRM/MRM in the verification step. The SRM/MRM data of ITGA5, GPRC5A, PDGFRB, and TFRC are shown. P, polyp. C, cancer without metastasis. Cm, cancer with metastasis. Area ratio, the ratio of the peak area of the endogenous peptide to that of the SI peptide.
Fig. 4.
Fig. 4.
Validation of the biomarker candidate C8orf55. A, The iTRAQ and SRM/MRM data of C8orf55. P, polyp. C, cancer without metastasis. Cm, cancer with metastasis. Area ratio, the ratio of the peak area of the endogenous peptide to that of the SI peptide. Assays were constructed to measure two distinct peptides per-protein listed in supplemental Table S5 and that the individual assays for each of the two peptides are labeled SRM-1 and SRM-2. B, Western blotting analysis of polyps and cancer with and without metastasis using an anti-C8orf55 antibody. C, Immunohistochemical staining of colorectal cancer tissue using an anti-C8orf55 antibody. Left panel: lower magnification. Right panel: higher magnification. The arrowhead shows areas of stained tumor cells and the arrow shows normal colon epithelial glands. Bar, 100 μm.
Fig. 5.
Fig. 5.
Tissue microarray of C8orf55. C8orf55 immunohistochemistry score (staining intensity) between normal and cancer tissues. Statistical analyses were performed using the Wilcoxon test.
See this image and copyright information in PMC

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