title: Trifluoroethanol-containing RP-HPLC mobile phases for the separation of transmembrane peptides human glycophorin-A, integrin alpha-1, and p24: analysis and prevention of potential side reactions due to formic acid.
abstract: Reversed-phase high-pressure liquid chromatography analysis and purification of three hydrophobic, aggregation-prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2-trifluoroethanol (TFE). The addition of 10-16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE-containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711: 181-186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM-peptide purification. Characterization of the potential side reactions using MALDI and ESI-LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE-formic acid possibly induces O-formylation of the Ser residue and N-formylation of the N-terminus of peptides. The conditions for selective removal of the formyl groups from TM peptides were also screened. We believe that these results will expand our ability to analyze and prepare hydrophobic, aggregation-prone TM peptides and proteins.
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan; JST ERATO, Lipid Active Structure Project, Osaka University, Toyonaka, Osaka, 560-0043, Japan; Project Research Centre for Fundamental Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Hara
authors.forename: Yue
authors.initials: Y
authors.lastname: Huang
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ito
authors.forename: Toru
authors.initials: T
authors.lastname: Kawakami
authors.forename: Hironobu
authors.initials: H
authors.lastname: Hojo
authors.forename: Michio
authors.initials: M
authors.lastname: Murata
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005561:Formates / Q000737:chemistry*; D006021:Glycophorins / Q000032:analysis*; D006801:Humans; D039423:Integrin alpha1 / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D014270:Trifluoroethanol / Q000737:chemistry*
chemical_list: D005561:Formates; D006021:Glycophorins; D039423:Integrin alpha1; D008565:Membrane Proteins; C030544:formic acid; D014270:Trifluoroethanol
keywords: 2,2,2-trifluoroethanol-containing mobile phase; HPLC analysis; deformylation; formylation; peptide purification; transmembrane peptide