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Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

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Abstract

Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate–polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.

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Acknowledgments

We thank Universiti Sains Malaysia for providing the Research University (RU) grant to carry out and in order to fund this project (Research Universiti Grant, grant no. 1001/PFARMASI/815007) and the Institute of Postgraduate Studies (IPS) for the financial support during the postgraduate study S.-R. Lim. We also want to extend our gratitude to the Ministry of Health in Malaysia for providing tissue specimens for the analysis. Last but not the least, we thank the National Institute of Nutraceutical and Pharmaceutical, Malaysia for their kindness in allowing us to use the LC/MS/MS.

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Authors and Affiliations

  1. School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia

    Shi-Rou Lim & Lay-Harn Gam

  2. Department of Surgery, Penang General Hospital, Penang, Malaysia

    Boon-Hui Gooi & Manjit Singh

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  1. Shi-Rou Lim
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  2. Boon-Hui Gooi
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Correspondence to Lay-Harn Gam.

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Lim, SR., Gooi, BH., Singh, M. et al. Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE. Appl Biochem Biotechnol 165, 1211–1224 (2011). https://doi.org/10.1007/s12010-011-9339-3

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