title: Protein microarrays using liquid phase fractionation of cell lysates.
abstract: We describe an approach in which protein microarrays are produced using a two-dimensional (2-D) liquid phase fractionation of cell lysates. The method involves a pI-based fractionation using chromatofocusing in the first dimension followed by nonporous reversed-phase high-performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero-diagnostic markers for prostate cancer. Importantly, this method allows for use of post-translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.
authors.affiliation: Department of Chemistry, University of Michigan, 930 N. University Avenue, Ann Arbor, MI 48109-1055, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Yan
authors.forename: Arun
authors.initials: A
authors.lastname: Sreekumar
authors.forename: Bharathi
authors.initials: B
authors.lastname: Laxman
authors.forename: Arul M
authors.initials: AM
authors.lastname: Chinnaiyan
authors.forename: David M
authors.initials: DM
authors.lastname: Lubman
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Barder
mesh_terms: D001331:Automation; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003101:Collodion / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D011471:Prostatic Neoplasms / Q000175:diagnosis / Q000378:metabolism; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D011506:Proteins; D003101:Collodion