Combination of COFRADIC and high temperature-extended column length conventional liquid chromatography: a very efficient way to tackle complex protein samples, such as serum.
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.
Sandra
Koen
K
Department of Medical Protein Research and Biochemistry, Flanders Interuniversity Institute for Biotechnology and Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
Verleysen
Katleen
K
Labeur
Christine
C
Vanneste
Lies
L
D'Hondt
Filip
F
Thomas
Grégoire
G
Kas
Koen
K
Gevaert
Kris
K
Vandekerckhove
Joël
J
Sandra
Pat
P
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
D001798:Blood Proteins; D014357:Trypsin