Identification of Differentially Expressed Proteins in the Serum of Colorectal Cancer Patients Using 2D-DIGE Proteomics Analysis

Lay Cheng Lim, Mee Lee Looi, Syed Zulkifli Syed Zakaria, Ismail Sagap, Isa Mohammed Rose, Siok Fong Chin, A. Rahman A. Jamal

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Early detection of colorectal cancer (CRC) is vital for the improvement of disease prognosis. However to date there are no blood-based biomarkers sensitive and specific enough for early diagnosis. We analysed the differences in serum protein expression of early stage CRC (Dukes’ A and B) and late stage CRC (Dukes’ C and D) against normal controls using 2D Fluorescence Difference Gel Electrophoresis (2D-DIGE). Analysis of the 2D maps showed that 23 proteins were differentially expressed between groups (p ≤ 0.05) and these proteins were identified with LC-MS/MS. Eight proteins were up-regulated and 2 down-regulated in patients with early CRC, whereas 14 proteins were up-regulated and 4 down-regulated in those with late CRC compared to normal controls (p ≤ 0.05). Five proteins, namely apolipoprotein A1 (APOA1), apolipoprotein E (APOE), complement factor H (CFH), galectin-7 (GAL7) and synaptojanin-2 (SYNJ2) were validated using ELISA and only APOA1 and GAL-7 showed consistent findings. Further validation using immunohistochemistry showed negative immunoreactivity for GAL-7 in CRC tissues, suggesting that GAL-7 detected in the serum did not originate from the CRC tumour. APOA1 showed positive immunoreactivity but its expression did not correlate with Dukes’ staging (p = 0.314), tumour grading (p = 0.880) and lymph node involvement (p = 0.108). Differences in APOA1 isoforms and/or conformation between serum and tissue samples as well as tumour heterogeneity may explain for the discrepancies between DIGE and ELISA when compared to immunohistochemistry. Structural and functional studies of APOA1 in future would best describe the role of APOA1 in CRC.

Original languageEnglish
Pages (from-to)169-177
Number of pages9
JournalPathology and Oncology Research
Volume22
Issue number1
DOIs
Publication statusPublished - 1 Jan 2016

Fingerprint

Proteomics
Electrophoresis
Blood Proteins
Colorectal Neoplasms
Fluorescence
Gels
Apolipoprotein A-I
Proteins
Enzyme-Linked Immunosorbent Assay
Immunohistochemistry
Galectins
Complement Factor H
Neoplasm Grading
Apolipoproteins
Apolipoproteins E
Serum
Early Detection of Cancer
Early Diagnosis
Protein Isoforms
Biomarkers

Keywords

  • 2D-DIGE
  • Apolipoprotein A1
  • Colorectal cancer
  • LC-MS/MS
  • Proteomics

ASJC Scopus subject areas

  • Cancer Research
  • Oncology
  • Pathology and Forensic Medicine

Cite this

@article{c233b07501e04835b37be7b165b516bf,
title = "Identification of Differentially Expressed Proteins in the Serum of Colorectal Cancer Patients Using 2D-DIGE Proteomics Analysis",
abstract = "Early detection of colorectal cancer (CRC) is vital for the improvement of disease prognosis. However to date there are no blood-based biomarkers sensitive and specific enough for early diagnosis. We analysed the differences in serum protein expression of early stage CRC (Dukes’ A and B) and late stage CRC (Dukes’ C and D) against normal controls using 2D Fluorescence Difference Gel Electrophoresis (2D-DIGE). Analysis of the 2D maps showed that 23 proteins were differentially expressed between groups (p ≤ 0.05) and these proteins were identified with LC-MS/MS. Eight proteins were up-regulated and 2 down-regulated in patients with early CRC, whereas 14 proteins were up-regulated and 4 down-regulated in those with late CRC compared to normal controls (p ≤ 0.05). Five proteins, namely apolipoprotein A1 (APOA1), apolipoprotein E (APOE), complement factor H (CFH), galectin-7 (GAL7) and synaptojanin-2 (SYNJ2) were validated using ELISA and only APOA1 and GAL-7 showed consistent findings. Further validation using immunohistochemistry showed negative immunoreactivity for GAL-7 in CRC tissues, suggesting that GAL-7 detected in the serum did not originate from the CRC tumour. APOA1 showed positive immunoreactivity but its expression did not correlate with Dukes’ staging (p = 0.314), tumour grading (p = 0.880) and lymph node involvement (p = 0.108). Differences in APOA1 isoforms and/or conformation between serum and tissue samples as well as tumour heterogeneity may explain for the discrepancies between DIGE and ELISA when compared to immunohistochemistry. Structural and functional studies of APOA1 in future would best describe the role of APOA1 in CRC.",
keywords = "2D-DIGE, Apolipoprotein A1, Colorectal cancer, LC-MS/MS, Proteomics",
author = "Lim, {Lay Cheng} and Looi, {Mee Lee} and {Syed Zakaria}, {Syed Zulkifli} and Ismail Sagap and Rose, {Isa Mohammed} and Chin, {Siok Fong} and {A. Jamal}, {A. Rahman}",
year = "2016",
month = "1",
day = "1",
doi = "10.1007/s12253-015-9991-y",
language = "English",
volume = "22",
pages = "169--177",
journal = "Pathology and Oncology Research",
issn = "1219-4956",
publisher = "Springer Netherlands",
number = "1",

}

TY - JOUR

T1 - Identification of Differentially Expressed Proteins in the Serum of Colorectal Cancer Patients Using 2D-DIGE Proteomics Analysis

AU - Lim, Lay Cheng

AU - Looi, Mee Lee

AU - Syed Zakaria, Syed Zulkifli

AU - Sagap, Ismail

AU - Rose, Isa Mohammed

AU - Chin, Siok Fong

AU - A. Jamal, A. Rahman

PY - 2016/1/1

Y1 - 2016/1/1

N2 - Early detection of colorectal cancer (CRC) is vital for the improvement of disease prognosis. However to date there are no blood-based biomarkers sensitive and specific enough for early diagnosis. We analysed the differences in serum protein expression of early stage CRC (Dukes’ A and B) and late stage CRC (Dukes’ C and D) against normal controls using 2D Fluorescence Difference Gel Electrophoresis (2D-DIGE). Analysis of the 2D maps showed that 23 proteins were differentially expressed between groups (p ≤ 0.05) and these proteins were identified with LC-MS/MS. Eight proteins were up-regulated and 2 down-regulated in patients with early CRC, whereas 14 proteins were up-regulated and 4 down-regulated in those with late CRC compared to normal controls (p ≤ 0.05). Five proteins, namely apolipoprotein A1 (APOA1), apolipoprotein E (APOE), complement factor H (CFH), galectin-7 (GAL7) and synaptojanin-2 (SYNJ2) were validated using ELISA and only APOA1 and GAL-7 showed consistent findings. Further validation using immunohistochemistry showed negative immunoreactivity for GAL-7 in CRC tissues, suggesting that GAL-7 detected in the serum did not originate from the CRC tumour. APOA1 showed positive immunoreactivity but its expression did not correlate with Dukes’ staging (p = 0.314), tumour grading (p = 0.880) and lymph node involvement (p = 0.108). Differences in APOA1 isoforms and/or conformation between serum and tissue samples as well as tumour heterogeneity may explain for the discrepancies between DIGE and ELISA when compared to immunohistochemistry. Structural and functional studies of APOA1 in future would best describe the role of APOA1 in CRC.

AB - Early detection of colorectal cancer (CRC) is vital for the improvement of disease prognosis. However to date there are no blood-based biomarkers sensitive and specific enough for early diagnosis. We analysed the differences in serum protein expression of early stage CRC (Dukes’ A and B) and late stage CRC (Dukes’ C and D) against normal controls using 2D Fluorescence Difference Gel Electrophoresis (2D-DIGE). Analysis of the 2D maps showed that 23 proteins were differentially expressed between groups (p ≤ 0.05) and these proteins were identified with LC-MS/MS. Eight proteins were up-regulated and 2 down-regulated in patients with early CRC, whereas 14 proteins were up-regulated and 4 down-regulated in those with late CRC compared to normal controls (p ≤ 0.05). Five proteins, namely apolipoprotein A1 (APOA1), apolipoprotein E (APOE), complement factor H (CFH), galectin-7 (GAL7) and synaptojanin-2 (SYNJ2) were validated using ELISA and only APOA1 and GAL-7 showed consistent findings. Further validation using immunohistochemistry showed negative immunoreactivity for GAL-7 in CRC tissues, suggesting that GAL-7 detected in the serum did not originate from the CRC tumour. APOA1 showed positive immunoreactivity but its expression did not correlate with Dukes’ staging (p = 0.314), tumour grading (p = 0.880) and lymph node involvement (p = 0.108). Differences in APOA1 isoforms and/or conformation between serum and tissue samples as well as tumour heterogeneity may explain for the discrepancies between DIGE and ELISA when compared to immunohistochemistry. Structural and functional studies of APOA1 in future would best describe the role of APOA1 in CRC.

KW - 2D-DIGE

KW - Apolipoprotein A1

KW - Colorectal cancer

KW - LC-MS/MS

KW - Proteomics

UR - http://www.scopus.com/inward/record.url?scp=84951573963&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84951573963&partnerID=8YFLogxK

U2 - 10.1007/s12253-015-9991-y

DO - 10.1007/s12253-015-9991-y

M3 - Article

C2 - 26463353

AN - SCOPUS:84951573963

VL - 22

SP - 169

EP - 177

JO - Pathology and Oncology Research

JF - Pathology and Oncology Research

SN - 1219-4956

IS - 1

ER -