Separation techniques and mass spectrometry
for analysis of bioactive components
Blanka Hégrová
Department of Chemistry, Faculty of Science, Masaryk University, Kotlá ská 2, 611 37 Brno,
Czech Republic, blanka.hegrova@seznam.cz
Abstract
Analysis of biological active compounds comprises large number of complex, which trench
on many areas of the human live. Bioactive compounds are any compounds, which influence
significantly organisms. We used separation techniques and mass spectrometry for
determination of specific bioactive compounds, namely antifungal proteins from barley/malt
kernels and plant sterols.
This research is focused on relatively small cationic and amphipatic PR peptides, mainly
thionins, and nsLTPs, incident in barley grains. These peptides were extracted by various
solvents from barley and malt kernels. One-dimensional SDS PAGE and MALDI MS were
used for their detection. SDS PAGE and MALDI MS were applied as fast screening tools for
observing the relationship between gushing and peptides/proteins. In addition, changes to the
proteins expression reflecting the quality of barley and malt kernels, dependent on the variety
and conditions of pathogen treatment, for 20 samples originating from a single fast station and
the same preceding crop were studied. Finally, it was found by both SDS PAGE and MALDI
MS that the presence of basic peptides, presumably hordothionins and non-specific lipid
transfer protein type 1, did not correlate with the degree of gushing for malt (|r| 0.07,
0.34 ), (|r| 0.01, 0.49 ), respectively.
In second application, a new methodology for chromatographic detection of nonpolar
compounds is provided. MALDI MS is a routinely used technique for the analysis of large
polar nonvolatile molecules, such as biopolymers and synthetic polymers. However,
nonconventional matrices, such as silver ions form readily adducts with sterols, which allows
sensitive MS analysis of these nonpolar analytes in positive mode. The MALDI MS detection
limit of sterols (S/N = 3) with silver nanoparticles as matrix were about 10 femtomoles per
spot. Liquid chromatography of sterols was optimised for their fraction collection and
subsequent MALDI MS determination using silver nanoparticles as matrix. Appropriate
separation of sterols was obtained in less than 9 minutes on Luna C8 column (150x4.6 mm
I.D., 3 m) with MetOH:water (97:3) as eluent. The experiments were performed on HPLC
system with UV optical detection. After splitter, the fractions were collected on MALDI
target. The LC effluent was deposited as 4-second fractions with 500 nanoliter drops. MALDI
MS signal of separated sterols (~ 15 picomoles injected, splitter 100:1) was two to three
orders above LOD determined by MALDI MS of sterol standards. The presented off-line LC
­ MALDI MS is a viable alternative to GC ­ MS for analysis of nonpolar compounds.