title: Identification and quantification of basic and acidic proteins using solution-based two-dimensional protein fractionation and label-free or 18O-labeling mass spectrometry.
abstract: Reduction in sample complexity enables more thorough proteomic analysis using mass spectrometry (MS). A solution-based two-dimensional (2D) protein fractionation system, ProteomeLab PF 2D, has recently become available for sample fractionation and complexity reduction. PF 2D resolves proteins by isoelectric point (pI) and hydrophobicity in the first and second dimensions, respectively. It offers distinctive advantages over 2D gel electrophoresis with respects to automation of the fractionation processes and characterization of proteins having extreme pIs. Besides fractionation, PF 2D is equipped with built-in UV detectors intended for relative quantification of proteins in contrasting samples using its software tools. In this study, we utilized PF 2D for the identification of basic and acidic proteins in mammalian cells, which are generally under-characterized. In addition, mass spectrometric methods (label-free and 18O-labeling) were employed to complement protein quantification based on UV absorbance. Our studies indicate that the selection of chromatographic fractions could impact protein identification and that the UV-based quantification for contrasting complex proteomes is constrained by coelution or partial coelution of proteins. In contrast, the quantification post PF 2D chromatography based on label-free or 18O-labeling mass spectrometry provides an alternative platform for basic/acidic protein identification and quantification. With the use of HCT116 colon carcinoma cells, a total of 305 basic and 183 acidic proteins was identified. Quantitative proteomics revealed that 17 of these proteins were differentially expressed in HCT116 p53-/- cells.
authors.affiliation: Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Wells W
authors.initials: WW
authors.lastname: Wu
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wang
authors.forename: Ming-Jiun
authors.initials: MJ
authors.lastname: Yu
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
authors.forename: Rong-Fong
authors.initials: RF
authors.lastname: Shen
mesh_terms: D000143:Acids / Q000737:chemistry; D000468:Alkalies / Q000737:chemistry; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000295:instrumentation*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007526:Isoelectric Point; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D010103:Oxygen Isotopes / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
chemical_list: D000143:Acids; D000468:Alkalies; D010103:Oxygen Isotopes; D011506:Proteins