Differential Screening and Mass Mapping of Proteins from Premalignant and Cancer Cell Lines Using Nonporous Reversed-Phase HPLC Coupled with Mass Spectrometric Analysis

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Abstract
Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-μm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30−40 μg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approximately 75−80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual “1-D gel” and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

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