title: Improving the resolution of neuropeptides in rat brain with on-line HILIC-RP compared to on-line SCX-RP.
abstract: Our two already established on-line 2-D LC systems, a strong cation exchange-RP chromatography (SCX-RP) system and a hydrophilic interaction LC (HILIC)-RP 2-D LC system, were compared to explore which system is best suited for our further studies of differences in cerebral neuropeptide expression as a function of hypoxia-caused stress. The same mass spectrometer and database search parameters were applied in both systems. In total, 19 first dimension fractions were collected with the novel on-line HILIC-RP system, including a Hypercarb SPE column that was applied to trap the compounds not retained on a Kromasil C18 enrichment column. In contrast, six fractions were collected in the SCX-RP method, due to practical limitations of this traditional on-line 2-D LC system. With the on-line HILIC-RP system three times more peaks were detected. It was observed that most of the compounds eluted in the first two fractions in the SCX-RP method, while in the 2-D HILIC-RP method there seemed to be no correlation between peaks detected and fraction number. Thus, from this systematic study it seems that on-line HILIC-RP chromatography is the method of choice for comparative peptidomics of cerebral neuropeptides in future studies.
authors.affiliation: Department of Chemistry, University of Oslo, Blindern, Oslo, Norway.
authors.forename: Albena
authors.initials: A
authors.lastname: Mihailova
authors.forename: Helle
authors.initials: H
authors.lastname: Malerød
authors.forename: Steven Ray
authors.initials: SR
authors.lastname: Wilson
authors.forename: Bartosz
authors.initials: B
authors.lastname: Karaszewski
authors.forename: Roman
authors.initials: R
authors.lastname: Hauser
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
authors.forename: Tyge
authors.initials: T
authors.lastname: Greibrokk
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D009479:Neuropeptides / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D009479:Neuropeptides