title: On-Line multitasking analytical proteomics: how to separate, reduce, alkylate and digest whole proteins in an on-Line multidimensional chromatography system coupled to MS.
abstract: An on-Line multidimensional system has been developed, consisting of pH gradient strong anion exchange chromatography of native proteins in the first dimension with subsequent trapping and on-column reduction/alkylation on C4 trap columns and RP separation of the alkylated proteins in the second dimension followed by on-column tryptic digestion and electrospray MS detection. The system was evaluated using model proteins and a human urine sample. Compared to the commonly used in-solution alkylation method, the developed on-column method provides an equivalent efficiency. The recovery from the C4 trap columns of the alkylated proteins relative to the native state was from 94 to 102%. On-column tryptic digestion was satisfactory for many, but not for all proteins. The whole analytical procedure was performed on-Line with packed capillary columns for a total time of 320 min for the first ion exchange fraction, with additional 60 min for each subsequent fraction.
authors.affiliation: Department of Chemistry, University of Oslo, Blindern, Oslo, Norway. b.q.tran@kjemi.uio.no
authors.forename: Bao Quoc
authors.initials: BQ
authors.lastname: Tran
authors.forename: Håvard
authors.initials: H
authors.lastname: Loftheim
authors.forename: Leon
authors.initials: L
authors.lastname: Reubsaet
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
authors.forename: Tyge
authors.initials: T
authors.lastname: Greibrokk
mesh_terms: D000478:Alkylation; D000818:Animals; D002417:Cattle; D002645:Chickens; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D045304:Cytochromes c / Q000032:analysis; D004800:Enzymes, Immobilized / Q000737:chemistry; D006736:Horses; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000032:analysis; D009113:Muramidase / Q000032:analysis; D009211:Myoglobin / Q000032:analysis; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin / Q000737:chemistry; D014556:Urine / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D007782:Lactoglobulins; D009211:Myoglobin; D011506:Proteins; D012709:Serum Albumin; D045304:Cytochromes c; D009113:Muramidase; D014357:Trypsin