2200 J. Sep. Sci. 2014, 37, 2200–2207 Shah Hussain 1 Y¨ uksel G ¨ uzel 1 Cornelia Pezzei 1 Matthias Rainer 1 Christian W. Huck 1 G¨ unther K. Bonn 1,2 1 Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain, Innsbruck, Austria 2 ADSI-Austrian Drug Screening Institute, Innrain, Innsbruck, Austria Received April 7, 2014 Revised May 30, 2014 Accepted May 31, 2014 Research Article Solid-phase extraction of plant thionins employing aluminum silicate based extraction columns Thionins belong to a family of cysteine-rich, low-molecular-weight (5 KDa) biologically active proteins in the plant kingdom. They display a broad cellular toxicity against a wide range of organisms and eukaryotic cell lines. Thionins protect plants against different pathogens, including bacteria and fungi. A highly selective solid-phase extraction method for plant thionins is reported deploying aluminum silicate (3:2 mullite) powder as a sorbent in extraction columns. Mullite was shown to considerably improve selectivity compared to a previously described zirconium silicate embedded poly(styrene-co-divinylbenzene) mono- lithic polymer. Due to the presence of aluminum(III), mullite offers electrostatic interactions for the selective isolation of cysteine-rich proteins. In comparison to zirconium(IV) silicate, aluminum(III) silicate showed reduced interactions towards proteins which resulted into superior washings of unspecific compounds while still retaining cysteine-rich thionins. In the presented study, European mistletoe, wheat and barley samples were subjected to solid-phase extraction analysis for isolation of viscotoxins, purothionins and hordothionins, respectively. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy was used for determining the selectivity of the sorbent toward thionins. The selectively retained thionins were quantified by colorimetric detection using the bicinchoninic acid assay. For peptide mass-fingerprint analysis tryptic digests of eluates were examined. Keywords: Hordothionins / Mullite / Purothionins / Solid-phase extraction / Vis- cotoxins DOI 10.1002/jssc.201400385  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction Thionins comprise an assembly of cysteine-rich, low- molecular-weight (5 KDa) basic proteins with a polypep- tide chain of 45–48 amino acids and 3–4 internal disulfide bridges. Thionins are customarily found in the endosperms of Gramineae, e.g. wheat and barley, as well as in diverse plant species, including leaves and stems (e.g. mistletoe, pyrularia, and rosids). They can be classified into two groups, namely /-thionins and -thionins (plant defesins). /-Thionins can be subdivided into the types I, II, III, IV, and V [1, 2]. Correspondence: Prof. Christian Huck, Head of Spectroscopy Group, Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck, Austria E-mail: Christian.W.Huck@uibk.ac.at Fax: +43 512 507 57399 Abbreviations: ACN, acetonitrile; BCA, bicinchoninic acid; DTT, dithiothreitol; FA, formic acid; HCCA, -cyano-4- hydroxycinnamic acid; IAA, iodoacetamide; nOGP, n-octyl -D-glucopyranoside; PBS, phosphate buffer saline; PSDZ, poly(styrene-co-divinylbenzene) embedded zirconium sili- cate; SA, sinapinic acid All five types of /-thionins seem to be exceedingly ho- mologous at the amino acid level. Primary structure determi- nations revealed that about 12–17% of the amino acids are cysteine residues. These cysteine residues are extremely con- served and are involved in disulphide bond formation [3, 4]. The crystal structure of crambin (type IV thionin) was the first to be resolved among thionins and was determined directly from anomalous scattering of sulfur. Crambin has the shape of the Greek capital letter gamma , the stem of gamma is an antiparallel pair of helices and the cross-arm comprises two antiparallel -strands, an irregular strand and a classic -turn [5]. A number of X-ray structure determinations of dif- ferent thionins, along with NMR studies clearly demonstrated a distinct architectural feature for these small proteins. Re- gardless of small variations in length (45–48 amino acids) /-thionins share similar three-dimensional structure with minute exceptions [1]. Type I thionins are found in the endosperm of grains, e.g. wheat and barley [1, 6] (-, -purothionins, and hordothion- ins, respectively). These highly basic proteins encompass 45 Colour Online: See the article online to view Figs. 1, 2, and 3 in colour. C  2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com