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Comparison of Strong Cation Exchange and SDS-PAGE Fractionation for Analysis of Multiprotein Complexes

  1. Author:
    Das, S.
    Bosley, A. D.
    Ye, X. Y.
    Chan, K. C.
    Chu, I.
    Green, J. E.
    Issaq, H. J.
    Veenstra, T. D.
    Andresson, T.

  2. Author Address

    [Das, Sudipto; Bosley, Allen D.; Ye, Xiaoying; Chan, King C.; Issaq, Haleem J.; Veenstra, Timothy D.; Andresson, Thorkell] SAIC Frederick Inc, Adv Technol Program, Lab Prote & Analyt Technol, NCI Frederick, Frederick, MD 21702 USA. [Chu, Isabel; Green, Jeffery E.] NCI, Lab Canc Biol & Genet, NIH, Bethesda, MD 20892 USA.;Andresson, T, SAIC Frederick Inc, Adv Technol Program, Lab Prote & Analyt Technol, NCI Frederick, Frederick, MD 21702 USA.
    1. Year: 2010
    2. Date: Dec
  2. Journal: Journal of Proteome Research
    1. 9
    2. 12
    3. Pages: 6696-6704
  4. Type of Article: Article
  5. ISSN: 1535-3893
  1. Abstract:

    Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX) The two complexes analyzed focused on the nuclear proteins Bmi 1 and GATA3 that were expressed within the cells at low and high levels respectively Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3 fold more proteins compared to separation at the protein level using SDS-PAGE The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage particularly for target proteins expressed at low levels

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External Sources

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  2. DOI: 10.1021/pr100843x
  3. View record in Web of Science®
  4. WOS: 000284856200052

Library Notes

  1. Fiscal Year: FY2010-2011
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