A two‐dimensional liquid‐phase separation scheme coupled with mass spectrometry (MS) is presented for proteomic analysis of cell lysates from normal and malignant breast epithelial cell lines. Liquid‐phase separations consist of isoelectric focusing as the first dimension and nonporous silica reverse‐phase high‐performance liquid chromatography (NPS‐RP‐HPLC) as the second dimension. Protein quantitation and mass measurement are performed using electrospray ionization‐time of flight MS (ESI‐TOF MS). Proteins are identified by peptide mass fingerprinting using matrix‐assisted laser desorption ionization‐time of flight MS (MALDI‐TOF MS) and MALDI‐quadrupole time of flight (QTOF)‐tandem mass spectrometry (MS/MS). Two pH regions with 50–60 unique proteins in each pH range were chosen for analysis. Mass maps were created that allowed visualization of protein quantitation differences between normal and malignant breast epithelial cells. Of the approximately 110 unique proteins observed from mass mapping experiments over the limited pH range, 40 (36%) were positively identified by peptide mass fingerprinting and assigned to bands in the mass maps. Of these 40 proteins, 22 were more highly expressed in one or more of the malignant cell lines. These proteins represent potential breast cancer biomarkers that could aid in diagnosis, therapy, or drug development.

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Background: "Chaperones" may enhance mutant enzyme activities, but therapeutic levels have not been shown in vivo. Results: A chaperone, isofagomine, stabilizes wild type and mutant acid ␤-glucosidases in tissues and sera and reduces visceral substrates in vivo. Conclusion: These effects are enhanced pre-versus postsynthetically. Significance: The results are proof of principle for the potential therapeutic use in residual enzyme diseases.

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-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg 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, approxinately 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.