Improving the resolution of neuropeptides in rat brain with on-line HILIC-RP compared to on-line SCX-RP.
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.
Mihailova
Albena
A
Department of Chemistry, University of Oslo, Blindern, Oslo, Norway.
Malerød
Helle
H
Wilson
Steven Ray
SR
Karaszewski
Bartosz
B
Hauser
Roman
R
Lundanes
Elsa
E
Greibrokk
Tyge
T
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
D009479:Neuropeptides