title: Liquid chromatographic separation of proteins derivatized with a fluorogenic reagent at cysteinyl residues on a non-porous column for differential proteomics analysis.
abstract: A wide-pore (30 nm) reversed-phase column (Intrada WP-RP, particle size 3 μm) was recently utilized for protein separation in differential proteomics analysis with fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS), and exerted a tremendous effect on finding biomarkers (e.g., for breast cancer). Further high-performance separation is required for highly complex protein mixtures. A recently prepared non-porous small-particle reversed-phase column (Presto FF-C18, particle size: 2 μm) was expected to more effectively separate derivatized protein mixtures than the wide-pore column. A preliminary experiment demonstrated that the peak capacity of the former was threefold greater than that of the latter in gradient elution of a fluorogenic derivatized model peptide, calcitonin. The FD-LC-MS/MS method with a non-porous column was then optimized and applied to separate liver mitochondrial proteins that were not efficiently separated with the wide-pore column. As a result, high-performance separation of mitochondrial proteins was accomplished, and differential proteomics analysis of liver mitochondrial proteins in a hepatitis-infected mouse model was achieved using the FD-LC-MS/MS method with the non-porous column. This result suggests the non-porous small-particle column as a replacement for the wide-pore column for differential proteomics analysis in the FD-LC-MS/MS method.
authors.affiliation: Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
authors.forename: Akiyo
authors.initials: A
authors.lastname: Koshiyama
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ichibangase
authors.forename: Kyoji
authors.initials: K
authors.lastname: Moriya
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Koike
authors.forename: Itaru
authors.initials: I
authors.lastname: Yazawa
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Imai
mesh_terms: D000818:Animals; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D003545:Cysteine / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006505:Hepatitis / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008930:Mitochondria, Liver / Q000737:chemistry / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010069:Oxadiazoles / Q000737:chemistry; D016062:Porosity; D040901:Proteomics / Q000379:methods*; D013449:Sulfonamides / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: C491139:4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole; D005456:Fluorescent Dyes; D024101:Mitochondrial Proteins; D010069:Oxadiazoles; D013449:Sulfonamides; D003545:Cysteine