Proteomics Analysis for Helicobacter pylori-infected Gastric Mucosa

Ho Suk Kang; Sung Noh Hong; Hye Rim Park; Mi Jung Kwon; Jun Haeng Lee; Jae J. Kim

doi:10.4166/kjg.2014.64.1.10

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Kang, Hong, Park, Kwon, Lee, and Kim: Proteomics Analysis for Helicobacter pylori-infected Gastric Mucosa

Original Article

Korean J Gastroenterol 2014;64(1):10-17.

Published online: 4 October 2014

DOI: https://doi.org/10.4166/kjg.2014.64.1.10

Proteomics Analysis for Helicobacter pylori-infected Gastric Mucosa

Ho Suk Kang¹, Sung Noh Hong³, Hye Rim Park², Mi Jung Kwon², Jun Haeng Lee³, Jae J. Kim³

¹Department of Internal Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea

²Department of Pathology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea

³Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Correspondence to: Sung Noh Hong, Division of Gastroenterology, Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 135-710, Korea. Tel: +82-2-3410-3409, Fax: +82-2-3410-3849, E-mail: gisnhong@gmail.com

Received 17 January 20147 May 2014 Accepted 16 May 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background/Aims

Helicobacter pylori infection is linked to the development of gastric cancer. H. pylori-associated gastric inflammation is considered to be the first important step in the histogenesis of such neoplasia. However, studies that compare proteome of gastric mucosa infected with or without H. pylori are lacking.

Methods

We employed proteomics analysis on the endoscopic biopsy specimens of gastric mucosa obtained from two groups (30 cases): healthy subjects without H. pylori infection (15 cases), and gastritis patients with H. pylori infection (15 cases). The pooled proteins obtained from gastric mucosa infected with or without H. pylori were separated by two-dimensional gel electrophoresis and analyzed by a computer-aided program. The altered protein expressions were then identified by mass spectrometry and validated by Western blotting and immunohistochemistry.

Results

On mass spectrometry using MALDI TOF™ Analyzer, the up-regulation of Keratin 1, ezrin, adenosine triphosphate (ATP) synthase subunit alpha mitochondrial isoform c, Keratin type I cytoskeletal 19, and Keratin type I cytoskeletal 9 were identified; in contrast, 71 kd heat shock cognate protein, ATP synthase subunit alpha mitochondrial precursor, and annexin IV were down-regulated. Among them, membrane cytoskeleton linker ezrin was validated using Western blot and immunohistochemistry.

Conclusions

Expression of ezrin was significantly different between the gastric mucosa with and without H. pylori infection. Therefore, ezrin could be considered a promising potential molecular marker for detecting H. pylori infection in gastric mucosa.

Keywords: Hlicobacter pylori, Proteomics, Ezrin, Stomach neoplasms, Gastritis

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Fig. 1.

Two-dimensional gel electrophoresis profiles of Helicobacter pylori(−) stomach and H. pylori(＋) nodular gastritis. A total of 100  g of whole-cell protein from each strain was applied on 170-mm immobilized pH gradient strips with a range of pH 3–10, followed by 12% sodium dodecyl sulfate (SDS-PAGE), and visualized by silver staining (up-regulated 5 protein spots [round] and down-regulated 5 protein spots [square] in H. pylori infected gastric mucosa are indicated at figure). IEF, isoelectric focusing.

Fig. 2.

Up- (A) and down (B)-regulated protein spots in Helicobacter pylori infected gastric mucosa of 2-dimensional gel electrophoresis map. Circles indicate the differentially expressed proteins whose expression level was at least more than two times higher than the other group. The expression level was determined by the relative spot volume of the proteins compared to the total amount of the protein in the gel, and is expressed as the percentage volume (right panel).

Fig. 3.

Western blot for ezrin in gastric mucosa infected with and without Helicobacter pylori.

Fig. 4.

Immunohistochemistry for ezrin in gastric mucosal infected with and without Helicobacter pylori (×100).

Table 1.

Up- and Down-regulated Proteins Analyzed with MALDI TOF ^TM Mass Spectrometry

	Spot no.	Accession no.	MOWSE score	Masses matched	Protein MW (Da)	Protein name： Human	Up-/Down-regulation fold
Up-regulated spot	237	gi\|55956899	87	16	62,255	Keratin 1 [Homo sapiens]	＋2.4
	283	gi\|46249758	168	36	69,313	Ezrin [Homo sapiens]	＋2.2
	535	gi\|4757810	130	22	54,574	ATP synthase subunit alpha mitochondrial isoform c [Homo sapiens]	＋4.0
	797	gi\|24234699	274	35	44,079	Keratin, type I cytoskeletal 19 [Homo sapiens]	＋2.3
	1,891	gi\|55956899	103	17	62,255	Keratin, type I cytoskeletal 9	＋2.9
Down-regulated spot	365	gi\|5729877	91	21	71,082	71 kd heat shock cognate protein [Homo sapiens]	−4.2
	537	gi\|4757810	75	15	59,828	ATP synthase subunit alpha mitochondrial precursor	−4.2
	973	gi\|544492	43	1	62,681	Lymphoid-restricted membrane protein [Homo sapiens]	−2.8
	1,094	gi\|39645467	109	20	36,290	Annexin IV (placental anticoagulant protein II) [Homo sapiens]	−4.1
	1,560	gi\|117938314	66	11	21,023	Unnamed protein product [Homo sapiens]	−3.0

MALDI-TOF^TM (Applied Biosystems, Foster City, CA, USA).

MOWSE, molecular weight search; MW, molecula weight; ATP, adenosine triphosphate.

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