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Combined solid-phase extraction and 2D LC–MS for characterization of the neuropeptides in rat-brain tissue

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Abstract

A comprehensive two-dimensional capillary liquid chromatographic (2D LC) method has been established for determination of neuropeptides in rat brain tissue. Rats were exposed to different levels of stress before sacrificing and the aim of this study was to design a powerful separation and detection technique capable of characterizing differences between cerebral neuropeptide expression as a function of stress level. Rat brain samples were homogenized and subjected to clean-up by solid-phase extraction (SPE) on both a reversed-phase (C18) and a weak cation-exchange (CBA) cartridge. The samples were divided in two fractions (A and B) depending on retention on the CBA column. Subsequently, 50 μL of the sample were injected on to a strong cation exchanger (SCX) at a mobile phase pH of 3, which enabled preconcentration of positively charged compounds. The trapped compounds were eluted using step gradients of ammonium formate in water–ACN (90:10, v/v). Before enrichment in the second dimension, the eluate from the first dimension was diluted with water containing 0.1% TFA. The compounds eluting from the first dimension were trapped in the second dimension using a dual precolumn system consisting of two short capillary columns packed with Kromasil C18, 10 μm particles. Subsequently, the trapped compounds were backflushed on to a 10 cm long, 320 μm I.D. analytical column packed with Kromasil C18 3.5 μm particles, on which they were efficiently separated. Detection was performed using an ion-trap mass spectrometer (ITMS) in both the MS and the MS–MS mode. Comparison of base-peak chromatograms (BPC) from MS analysis of stressed and non-stressed rats clearly revealed several differences in neuropeptide expression. The MS–MS data obtained combined with Mascot software were employed for peptide identification.

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Authors and Affiliations

  1. Institute of Immunology, Rikshospitalet University Hospital, 0027, Oslo, Norway

    Anders Holm

  2. Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern 0315, Norway

    Espen Storbråten, Albena Mihailova, Bartosz Karaszewski, Elsa Lundanes & Tyge Greibrokk

  3. Department of Neurology of Adults, Medical University of Gdansk, 80-210 Gdansk, Poland

    Bartosz Karaszewski

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  1. Anders Holm
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  2. Espen Storbråten
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  3. Albena Mihailova
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  4. Bartosz Karaszewski
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  5. Elsa Lundanes
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Correspondence to Anders Holm.

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Holm, A., Storbråten, E., Mihailova, A. et al. Combined solid-phase extraction and 2D LC–MS for characterization of the neuropeptides in rat-brain tissue. Anal Bioanal Chem 382, 751–759 (2005). https://doi.org/10.1007/s00216-005-3146-z

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  • DOI: https://doi.org/10.1007/s00216-005-3146-z

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