| Research Article |
Open Access |
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| A Chloroform-Assisted Protein Isolation Method Followed by Capillary NanoLC-MS Identify Estrogen-Regulated Proteins from MCF7 Cells |
| Adaikkalam Vellaichamy1,a, Chin-Yo Lin1, Thin Thin Aye1, Govindarajan R. Kunde1, Alexey I. Nesvizhskii2, Edison T. Liu1, Siu Kwan Sze1,b* |
| 1Genome Institute of Singapore, 60 Biopolis Street, Singapore, 138672 |
| 2Department of Pathology, University of Michigan, MI 48109, USA |
| Present address: |
| aInstitute for Genomic Biology, UIUC, Urbana, IL 61801, USA |
| bSchool of Biological Sciences, Nanyang Technological University, Singapore |
| *Corresponding author: |
Siu Kwan Sze, PhD
Assistant Professor
School of
Biological Sciences
Nanyang Technological University
Singapore 637551
Tel: +65-6316-2852
Fax: +65-6791-3856
E-mail: sksze@ntu.edu.sg |
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| Received June 12, 2010; Accepted June 29, 2010; Published June 29, 2010 |
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| Citation: Vellaichamy A, Lin CY, Aye TT, Kunde GR, Nesvizhskii AI, et al. (2010) A Chloroform-Assisted Protein Isolation Method Followed by Capillary NanoLC-MS Identify Estrogen-Regulated Proteins from MCF7 Cells. J Proteomics Bioinform 3: 212-220. doi:10.4172/jpb.1000142 |
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| Copyright: © 2010 Vellaichamy A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
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| Abstract |
| Most commonly reported non-commercial protein isolation methods require the use of detergents and/or chaotropes for better yield, and they all need additional Purification or clean-up steps for subsequent mass spectrometry analysis. Moreover, there is no simple procedure available for obtaining both soluble and membrane proteins from the same sample. Here we describe a simple and detergent-free chloroform-assisted protein isolation (ChlAPI) method for mammalian cells and tissues, and demonstrated its suitability to mass spectrometry based proteome analysis. In this single-step method, cultured cells or grounded tissue were mixed in 10% chloroform in ammonium bicarbonate buffer to separate whole cell proteome into biphasic layers. Total number of aqueous phase proteins, as assessed by 2DE, was comparable to the proteins isolated with commonly used detergent containing buffer. Shotgun proteomics analysis of the aqueous and organic phase proteome fractions of MCF7 cells by LC-MS/MS resulted in Identification of a total of 752 and 593 proteins, respectively from IPI human protein database. Among the total of 1134 distinct and nonredundant proteins, 29.5% were predicted to be membrane localized; 78% of them wereidentified from organic phase fraction. Application of this new procedure to estrogen-treated MCF cells lead to the Identification of previously known as well as unknown estrogen-responsive gene products. These fi ndings suggest that the simple and inexpensive ChlAPI method described here is suitable for protein isolation from mammalian samples, and is readily compatible with 2DE and LC-MS/MS analyses. |
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