Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from who le cell lysates of human breast cell lines, The nonporous separation involv es the use of hard sphere silica beads of 1.5-mum diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa, Using onl y 30-40 mug of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mas s range, approximately 75-80 are more highly expressed, The molecular weigh t 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, Th e separated proteins can also be detected by UV absorption and differential ly expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MA LDI-TOF MS for identification. It is demonstrated that the expressed protei n profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant ca ncer cells to estradiol can be rapidly screened by this method, demonstrati ng significant changes in response to an external agent. Ultimately, the pr oteins can be studied by peptide mapping to search for posttranslational mo difications of the oncoproteins accompanying progression.