Profiling the progression of cancer: separation of microsomal proteins in MCF10 breast epithelial cell lines using nonporous chromatophoresis.
The heterogeneity of cellular protein expression has stimulated development of separations targeting smaller groups of related proteins rather than entire proteomes. The following work describes the development of a technique for the characterization of membrane subproteomes from five different breast epithelial cell lines. Intact membrane proteins are separated by hydrophobicity in the first dimension using nonporous reversed-phase high-performance liquid chromatography (RP-HPLC) to generate unique chromatographic profiles. Fractions of eluent are further separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to create distinct banding patterns. This hybrid liquid phase/gel phase method circumvents issues of membrane protein precipitation and provides a simple strategy aimed at isolating and characterizing a traditionally underrepresented protein class. Membrane protein profiles are created that discriminate between microsomal fractions of breast epithelial cells in different stages of neoplastic progression. Proteins are subsequently identified using matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) mass fingerprinting and MALDI-quadrupole time of flight - tandem mass spectrometry (QTOF-MS/MS) peptide sequencing. Furthermore, as this strategy preserves intact protein structure, further characterization can be performed on proteins producing mass fingerprint spectra and fragmentation spectra that did not result in database protein identifications. The coupling of nonporous RP-HPLC with SDS-PAGE provides a useful alternative to two-dimensional PAGE (2-D-PAGE) for membrane protein analysis.
O'Neil
Kimberly A
KA
Department of Chemistry, The University of Michigan, 930 N. University Avenue, Ann Arbor, MI 48109-1055, USA.
Miller
Fred R
FR
Barder
Timothy J
TJ
Lubman
David M
DM
D001940:Breast / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D018450:Disease Progression; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D014357:Trypsin