Toward high sequence coverage of proteins in human breast cancer cells using on-line monolith-based HPLC-ESI-TOF MS compared to CE MS

Abstract

A method is developed toward high sequence coverage of proteins isolated from human breast cancer MCF10 cell lines using a 2-D liquid separations. Monolithic-capillary columns prepared by copolymerizing styrene with divinylbenzene are used to achieve high-resolution separation of peptides from protein digests. This separation is performed with minimal sample preparation directly from the 2-D liquid fractionation of the cell lysate. The monolithic column separation is directly interfaced to ESI-TOF 14MS to obtain a peptide map. The protein digests were also analyzed by MALDI-TOF 14MS and an accurate M r of the intact protein was obtained using an HPLC-ESI-TOF 14MS. The result is that these techniques provide complementary information where nearly complete sequence coverage of the protein is obtained and can be compared to the experimental M r value. The high sequence coverage provides information on isoforms and other post-translational modifications that would not be available from methods that result in low sequence coverage. The results from the use of monolithic columns are compared to that obtained by CE-MS. The monolithic column separations provide a rugged and highly reproducible method for separating protein digests prior to MS analysis and is suited to confidently identify biomarkers associated with cancer progression.