title: Combination of COFRADIC and high temperature-extended column length conventional liquid chromatography: a very efficient way to tackle complex protein samples, such as serum.
abstract: The previously reported COmbined FRActional DIagonal Chromatography (COFRA-DIC) methodology, in which a subset of peptides representative for their parent proteins are sorted, is particularly powerful for whole proteome analysis. This peptide-centric technology is built around diagonal chromatography, where peptide separations are crucial. This paper presents high efficiency peptide separations, in which four 250 x 2.1 mm, 5 microm Zorbax 300SB-C18 columns (total length 1 m) were coupled at operating temperatures of 60'C using a dedicated LC oven and conventional LC equipment. The high efficiency separations were combined with the COFRADIC procedure. This extremely powerful combination resulted, for the analysis of serum, in an increase in the uniquely identified peptide sequences by a factor of 2.6, compared to the COFRADIC procedure on a 25 cm column. This is a reflection of the increased peak capacity obtained on the 1 m column, which was calculated to be a factor 2.7 higher than on the 25 cm column. Besides more efficient sorting, less ion suppression was noticed.
authors.affiliation: Department of Medical Protein Research and Biochemistry, Flanders Interuniversity Institute for Biotechnology and Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
authors.forename: Koen
authors.initials: K
authors.lastname: Sandra
authors.forename: Katleen
authors.initials: K
authors.lastname: Verleysen
authors.forename: Christine
authors.initials: C
authors.lastname: Labeur
authors.forename: Lies
authors.initials: L
authors.lastname: Vanneste
authors.forename: Filip
authors.initials: F
authors.lastname: D'Hondt
authors.forename: Grégoire
authors.initials: G
authors.lastname: Thomas
authors.forename: Koen
authors.initials: K
authors.lastname: Kas
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Pat
authors.initials: P
authors.lastname: Sandra
mesh_terms: D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry*; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D014357:Trypsin