title: High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis.
abstract: Orthogonal high-resolution separations are critical for attaining improved analytical dynamic range and protein coverage in proteomic measurements. High-pH reversed-phase liquid chromatography (RPLC), followed by fraction concatenation, affords better peptide analysis than conventional strong cation-exchange chromatography applied for 2D proteomic analysis. For example, concatenated high-pH RPLC increased identification of peptides (by 1.8-fold) and proteins (by 1.6-fold) in shotgun proteomics analyses of a digested human protein sample. Additional advantages of high-pH RPLC with fraction concatenation include improved protein sequence coverage, simplified sample processing and reduced sample losses, making this an attractive alternative to strong cation-exchange chromatography in conjunction with second-dimension low-pH RPLC for 2D proteomics analyses.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Shen
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000302:isolation & purification; D020543:Proteome / Q000302:isolation & purification*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; D020543:Proteome