Proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling

doi:10.1016/j.bbrc.2016.05.114

Biochemical and Biophysical Research Communications

Volume 476, Issue 4, 5 August 2016, Pages 286-292

https://doi.org/10.1016/j.bbrc.2016.05.114 Get rights and content

Highlights

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Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis.
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Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample.
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SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.

Abstract

Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP and NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses.

Introduction

Colorectal cancer (CRC) is the third or second most common type of malignancy in men or women, and is one of the leading cause of cancer-related mortality worldwide [1]. The mortality of CRC in China is on the rise dramatically in recent years. CRC has become the fifth most common cancer in males, and the third most common cancer in females [2]. Some molecular markers such as the carcino embryonic antigen (CEA) have been used in diagnosis CRC. However, these most commonly used diagnostic biomarkers do not provide sufficient sensitivity and specificity for early-stage CRC screening [3]. Although several novel CRC biomarkers have been proposed in recent years, none of them is approved for clinical applications. Hence, there is still an urgent need to identify new biomarkers that are capable of serving as diagnostic and prognostic markers for CRC.

Mass spectrometry-based proteomic technologies, such as two-dimensional difference gel electrophoresis (2D-DIGE) and stable isotope labeling have been used to discover new biomarkers in colorectal cancer. Unlike stable isotope labeling methods, label-free quantitation doesn’t need to introduce isotopes and can detect more proteins in a single experiment. In addition, many studies have shown that label-free quantitation can achieve similar accuracy as stable isotopic labeling methods [4], [5]. Zhou et al. [6] developed a simplified and user-friendly centrifugal proteomic reactor using a bench-top centrifuge for membrane protein identification from only 20 μg protein sample.

Herein, in order to discover potential biomarkers for CRC, we applied the centrifugal proteomic reactor coupled with label-free quantitative proteomics to compare the proteome between paired cancer mucosa and adjacent normal colorectal mucosa. A total of 2620 proteins were identified and quantified by label-free quantitation. Of which, 403 proteins were significantly changed (with P-value < 0.05) between CRC and normal tissues. Functional annotation was performed on analyzing the differentially expressed proteins to better understand the major events involved in CRC tumor-genesis. Three proteins (SOD3, PRELP, NGAL) were selected for further validation.

Section snippets

Materials

Acetonitrile and water for nano-HPLC were purchased from J.T. Baker (Phillipsburg, NJ). Trypsin was purchased from Promega (Madison,WI). Strong cation exchange (SCX) beads were obtained from Polymer Laboratories, Agilent, Inc. (Palo Alto, CA). CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, BP 571) and methanol were purchased from BioRad (Hercules, CA).

Tissue samples collection

Colonic biopsies were obtained from CRC patients by either colonoscopy or flexible sigmoidoscopy, followed by bowel

Proteome profiling biopsy samples from colorectal cancer and normal colonic mucosa

In order to compare the proteomes between colorectal cancer (cancer or C) and normal colonic mucosa (normal or N), 12 clinical biopsy samples originating from six patients were analyzed in this study (Fig. 1A, Materials and Supplementary Table S3). The total extracted proteins from the collected biopsy tissues were only among 30–50 μg. A simplified and user-friendly proteomic reactor was applied to process clinical samples. The resulting tryptic peptides were analyzed by LC-MS/MS on a linear

Acknowledgements

This study was supported by Grant No. 21375138 from National Natural Science Foundation of China, Grant No. 2013ZX09507001 from the Ministry of Science and Technology of China, “one hundred talent program” of Chinese Academy of Sciences and the Innovation Project of Instrument and Equipment Function Development (Grant No. YZ201542 etc.), the Bureau of Goods, Chinese Academy of Sciences, and Grant No. 31500110 from Natural Science for Youth Foundation of China, Grant No. 81322010 from National

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¹: These authors contributed equally to this work.

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Proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling

Highlights

Abstract

Introduction

Section snippets

Materials

Tissue samples collection

Proteome profiling biopsy samples from colorectal cancer and normal colonic mucosa

Acknowledgements

Cancer Genet. Cytogenet.

Biochem. Biophys. Res. Commun.

Lab. Investig.

Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012

Int. J. Cancer

Annual report on status of cancer in China, 2010

Chin. J. Cancer Res.

Linking patient outcome to high throughput protein expression data identifies novel regulators of colorectal adenocarcinoma aggressiveness

F1000Res

Extensive quantitative remodeling of the proteome between normal colon tissue and adenocarcinoma

Mol. Syst. Biol.

Itraq-based and label-free proteomics approaches for studies of human adenovirus infections

Int. J. Proteomics

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Mol. Cell Proteomics