Isolation, identification and quantification of differentially expressed proteins from cancerous and normal breast tissues

Ann Clin Biochem 2008;45:299-306
doi:10.1258/acb.2007.007104
© 2008 Association for Clinical Biochemistry

 

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Original Articles


Mohd Izani Othman1,
M I A Majid2,
Manjit Singh3,
Che Nin Man4 and
Gam Lay-Harn1


1 School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM Penang, Malaysia;
2 National Institute of Pharmaceutical and Nutraceutical Biotechnology, Ministry of Science, Technology and Innovation, 62662 Putrajaya, Malaysia;
3 Department of Surgery, Penang General Hospital, 10990 Penang, Malaysia;
4 National Poison Centre, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia


Corresponding author: Dr Lay-Harn Gam. Email: layharn{at}usm.my


Background: Infiltrating ductal carcinoma (IDCA) is the most common typeof breast cancer accounting for 85% of all invasive breast cancers.

Methods: Forty tissue specimens comprising 20 pairs of normal and cancerous tissues were analysed. The tissues were homogenized and proteins were extracted using phosphate buffer. The protein extracts from each pair of cancerous and normal tissue were separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the same gel. The protein profiles of both the tissues were compared, and the differentially expressed proteins that were detected at >70% in one or both of the tissue types were selected for protein identification analysis. Target proteins were excised and digested in situ with trypsin prior to liquidchromatography/tandem mass spectrometry (LC/MS/MS) analysis.A protein that was present in both tissue types was furtherquantified using extracted ions chromatogram.

Results: The proteins were grouped as down-regulated, up-regulated and unique proteins. Twenty-two proteins were identified and eight of the proteins were found unique to cancer. These proteins belong to various molecular classes, i.e. structural protein, hypothetical protein, cytoskeletal protein, enzyme, calcium binding protein and extracellular matrix protein. One extracellular matrix protein, namely collagen -1(I) chain precursor was foundunique to cancer. By virtue of its location on the cell surfaceand its function in cancer growth, this protein may be a biomarkercandidate for breast cancer.

Conclusions: The proteins identified in this study were present in at least70% of the tissues tested; therefore they should have significantroles in the development of IDCA.

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